Tsukune Aono is a painfully average high school freshman who is unable to get into any private schools due to his disappointing grades. The only school that he is finally accepted into turns out to be a secret school for supernatural monsters (yokai) who are in disguise as humans. The first student that Tsukune meets is the beautiful Moka Akashiya, who takes an immediate liking to him for an unusual reason; she is a vampire and is attracted to the sweet taste of his human blood. Tsukune quickly realizes that his admission into a monster school must have been a mistake and fears for his life.

Despite Moka's vampiric nature, she is a sweet girl and Tsukune quickly falls in love with her. He decides to stay at the monster boarding school to be near her despite the various dangers. He must hide his human identity from the teachers and other students while trying to keep his grades up and avoiding conflicts with the various not-so-mythological monsters lurking around campus. Tsukune soon discovers that when he removes the cross-shaped amulet attached to the rosary around Moka's neck, she transforms into her true form, a "super-vampire" that is stronger and more powerful than almost any other creature at the school. However, in super-vampire form, Moka has a different personality that is often cold and merciless, so the rosary amulet is only removed in dire emergencies. Tsukune and Moka soon make new friends as their stay at Yokai Academy progresses.

Tsukune Aono
Voiced by: Daisuke Kishio

Main male protagonist. He is a completely average, ordinary human boy who was accepted to the school by a mistake since humans are not permitted to enter. He is in love with Moka, who is his main motivation for staying at the school despite the danger it entails for him as a human. Tsukune's infatuation with Moka often results in him letting his guard down long enough for Moka to bite him on the neck and suck his blood; although he doesn't really mind in the end. Tsukune will do anything for Moka even if it means putting his life in danger. He is the only one that can take Moka's rosary off. He has been transformed into a temporary vampire on at least four occasions so far by Moka. However, she only does this when Tsukune's life is in grave danger, as Tsukune has made it clear that he does not want to be a vampire himself. As a sidenote, it is not the sucking of blood that causes this temporary vampirification. Rather, it is the injection of vampiric blood that causes the transformation. However, due to the constant injection of blood, Tsukune later transforms into a Ghoul and looses all control of himself. He now has to wear a special rosary, similar in fashion to Moka's, locked around his wrist to suppress his Ghoul powers and personality. Later, Tsukune is able to access his Ghoul power without losing control by 'breaking' a link on his rosary. However, once he has broken all the links, he will again become an out-of-control monster.

Moka Akashiya
Voiced by: Nana Mizuki

Moka is a student of the Yokai Private Academy. She's the first person Tsukune met when he first set foot on the monster realm. Moka's true form is a vampire but she is very sweet and kind. She takes a liking to Tsukune after sucking his blood and seems to get addicted on doing so. She later on starts to like Tsukune in a romantic way. They tried to kiss once, when it was Tsukune's birthday, but were interrupted. However, Moka also has another side. When her rosary around her neck is removed, the seal that was keeping her true nature is released. Thus Moka turns to a super vampire and has a personality completely opposite of the kind Moka. This Moka is cold and has a silver hair rather than pink. But, later on this Moka also takes a liking to Tsukune. One instance was when Tsukune threw himself in front of a monster to protect Moka. As a result, he got hurt and Moka (without the rosary) responded by saying "how dare you do this to Tsukune". She also injects him with some of her own blood when he was seriously wounded. Moka is very protective of Tsukune (she is like this in both forms). She gets jealous when another girl is with him, especially Kurono. Moka is also one of the most popular girls around the school so she gets a lot of attention around the guys, but she pays them no response with the exception of Tsukune (much to the guys' dismay). She ranks 13 on the school exams and also a member of the Newspaper Club. She's also afraid of water not because she can't swim, but because water has a paralyzing effect on vampires due to its purifying properties. After Tsukune transforms into a Ghoul, the awakened Moka is forced to fight him and almost kills him before an exorist arrives to intervene and suppress his rage. It's revealed in an omake that she loves tomato juice,but prefers Tsukune's blood more.

Yukari Sendo
Voiced by: Kimiko Koyama

Her true form is a witch. She always like wearing a witch costume (which consist of hat and cape), which is considered by some of the students to be a violation of the school rules. Despite being the smartest student around the Yokai Academy, ranking #1 on the examination, she was lonely and a serious prankster before meeting Moka. She initially hates Tsukune since she claims she's in love with Moka, and uses her magical powers to try and drive them apart. However, after both Tsukune and Moka save her from her monster classmates, she falls for HIM too. As a witch, she had serious problems with being discriminated against as the witches are viewed as the "border beings". Border beings are beings that are viewed as being something between humans and supernaturals as it is impossible to tell the difference by appearance alone. After making friends with Moka and Tsukune, she apologized to her class and became better received by her classmates. She's also the youngest of the students of the academy, being only 11 years old.

Kurumu Kurono
Voiced by: Misato Fukuen

Her true form is a succubus. She's quite jealous of Moka due to the fact that Moka's popularity is overshadowing her popularity. She tried to take Tsukune away from her by seducing and using her ability to charm people and make them her slave. Later on, Kurumu is only doing that because she wants to find her "destined one" among the guys to save her dying breed and has an assumption that Moka is blocking that path. She got defeated by the Super Vampire Moka and was spared because Tsukune persuaded the Super Vampire Moka to not kill her. She then makes a decision that Tsukune will be her "destined one" and was friends to Moka and Tsukune from then on. It is revealed in an omake that Kurono has a serious weakness for sweets-to the extent that she ate poisoned cookies that she made and planned to give to Moka.

Mizure Shirayuki
Voiced by: Rie Kugimiya

True form Yuki Onna "Ice Woman". Just like in Japanese myth, she has the ability to freeze and control ice at will. She also have the ability to make ice clones of herself. First appeared in ch. 18, she returned to school after a long abscence and showed an immediate interest in Tsukune. In the past, she once confessed to a teacher she liked, but the teacher tried to take advantage of her and she froze him. That and the rumors that spread after, made it hard for her to trust anyone. She took a liking to Tsukune because she believed that Tsukune was a loner just like herself. She believed that Moka was in her way of getting Tsukune's complete affection and thus tried to kill her. She eventually warmed up to the others after being saved by Tsukune in his temporary vampire form. After being saved she also starts stalking Tsukune on a daily basis.

Ruby Dojo
Voiced by: Saeko Chiba

A witch from the human realm, Ruby Dojo was part of a plot to destroy mankind, as a response to a large garbage dump that was polluting her master's sanctuary. Her hate for humans stems from a car accident that took her parents' lives. After receiving help from Tsukune and his friends from a fight, and remembering the kind of love her parents had given her, she attempts to persuade her master to call off her plan, but to no avail, as her master was bent on destroying the human world.

Ruby reappears, when Tsukune became out of control in his ghoul-state, and helps the exorcist in sealing his abilities. She now resides at Y?kai Academy, as the board chairman's aide.

In an omake, it reveals that the main reason for Ruby working under the board chairman is her deep crush on Tsukune and by working at the school she could be near him.

The anime omits the prescence of her master, drastically changing the events surrounding her.