"Rosario + Vampire" is set in a world where humans coexist with supernatural beings, though most humans are unaware of their existence. The story centers around Tsukune Aono, an ordinary human who accidentally enrolls in Yokai Academy, a school for monsters. Initially unaware of the school's true nature, Tsukune quickly discovers that all the students and faculty are supernatural creatures, including vampires, witches, werewolves, and other mythical beings. To survive, he must keep his human identity a secret, as humans are considered prey in this environment.

The narrative primarily revolves around Tsukune's relationships with the female students, particularly Moka Akashiya, a vampire who becomes his closest companion. Moka has a dual personality: her outer self is sweet and kind, while her inner self, revealed when her rosary seal is removed, is powerful, assertive, and fiercely protective of Tsukune. The rosary, which suppresses her true nature, is a key element of the story, as it symbolizes the balance between her two personas and her connection to Tsukune.

As the series progresses, Tsukune forms bonds with other female classmates, each representing a different supernatural species. These include Kurumu Kurono, a succubus; Yukari Sendo, a young witch; and Mizore Shirayuki, a snow woman. Together, they navigate the challenges of Yokai Academy, facing rival students, dangerous monsters, and external threats. The group often finds themselves in perilous situations, requiring them to rely on each other's unique abilities to overcome obstacles.

The plot explores themes of coexistence, identity, and acceptance, as Tsukune and his friends confront prejudice and discrimination within the monster world. The series balances comedic and lighthearted moments with action-packed sequences and occasional darker undertones. Tsukune's growth as a character is a central focus, as he evolves from a timid human into a more confident individual willing to protect his friends, even at great personal risk.

Throughout the series, the dynamics between the characters drive much of the narrative, with romantic tension and comedic misunderstandings adding to the interpersonal drama. The overarching plot involves uncovering secrets about Yokai Academy, the origins of Moka's rosary, and the true nature of the supernatural world. The story builds toward climactic confrontations with powerful adversaries, testing the bonds of friendship and loyalty among the main characters.