Tsukune Aono is a normal high school teenager, who is unable to get into any private school due to his poor grades. However, with the help from his parents, he gets enrolled to a private school called Yokai Academy. When he arrives at the school, he soon discovers that Yokai Academy is a secret private school for supernatural monsters and mythological creatures disguised as humans, and any human who finds out about its existence will eventually be killed without mercy. Realizing this problem, Tsukune fears for his life, thinking that his admission into Yokai Academy is a mistake. His luck soon changes when he quickly becomes friends with a beautiful girl Moka Akashiya, who turns out to be a vampire, attracted to the sweet taste of his blood, sometimes biting Tsukune on the neck when the chance seems right. While at the beginning of the series, Moka was upset upon discovering Tsukune's human identity, as she realizes that he is the first real friend she had ever made in her life. Despite Moka's vampiric nature, she is a kind girl and Tsukune quickly falls for her, and ultimately decides to stay at Yokai Academy, despite the various dangers while hiding his human identity from the faculty and other students. Tsukune soon discovers that when he removes the rosario around Moka's neck, she transforms into her true vampire form (her true perspective and personality is sealed, the sweet Moka being a different version or an artificial person), being stronger and more powerful than almost any other creature at the school, and Tsukune is the only person who can physically remove it in order for her to transform into her true self. However, "Inner Moka" has an entirely different personality, often cold and merciless, and thus the rosary is only removed in dire emergencies.

As the school year continues, Tsukune becomes involved with other students who start out as enemies, but who are won over into becoming his friends: Kurumu Kurono, a busty succubus; Ginei Morioka, a perverted lech of a werewolf who is a second-year student and president of the school's Newspaper Club; Ruby Tojo, a witch from the human world, Yukari Sendo an eleven-year-old witch; and Mizore Shirayuki, a yuki-onna (a teen version of the legendary snow woman of Japanese folk tales) who stalks Tsukune, believing them to be soulmates. However, after Tsukune befriends each of the girls, they become friendly rivals along with Moka, vying for Tsukune's affections, and later most become members of the school's Newspaper Club with Tsukune and stand by him even after they learn that he is really a human. Together they wind up fighting against a variety of enemies within the school body, be it students or teachers, who try to use their monstrous powers to bully or contol one or more of the group for their own selfish or destructive ends.

Later on in the second manga serialization, Kokoa Shuzen, Moka's younger half-sister, appears. And, as time goes on, they learn about the dangerous organization Fairy Tale, which seeks to destroy the human world and must do battle against them to keep the school and both the monster and human worlds safe; Tsukune also starts to undergo changes that truly solidify his role as the link between the human and monster worlds. Through body modification, Tsukune gains the ability to wield sorcery and control the vampire blood Moka gave him, but the secret of this blood may still cost Tsukune his humanity if it goes completely out of control.

Eventually, it is revealed that the Three Dark Lords [Akasha Blooodriver (Moka's mother), Tohofuhai and Tenmei Mikogami (Yokai Academy's headmaster]) defeated and sealed a powerful monster named Alucard, which tried to destroy the world 200 years ago and shared the same bloodline as Moka and her mother. The rosario is also revealed to seal Moka's true personality and power away (meaning Outer Moka is artificial), in order to keep Alucard contained. In order to awaken him, Fairy Tale, under Gyokuro Shuzen's (Moka's stepmother) command, kidnaps Moka. During the Newspaper club's attack on Fairy Tale HQ in an attempt to rescue her, the rosario's final secret is revealed; it's meant to control Alucard if he ever awakens. However, Tohofuhai later reveals the rosario actually synchronises with the essence of Akasha inside Alucard, with the end result being Gyokuro Shuzen's demise. At that moment, the Masked King (Fairy Tale's true leader) appears before the Newspaper Club, revealing himself to be Miyabi Fujisaki, who in turn is a clone of Alucard's original vampiric appearance, created shortly before he was sealed. He proceeds to reveal that he manipulated both sides to do battle with one another, in order to access whether the Rosario could control his main body.