"Maria Holic" centers on Kanako Miyamae, a second-year high school student who transfers to the prestigious all-girls Ame no Kisaki Catholic School. Kanako has a deep-seated fear of men due to traumatic childhood experiences, which has led her to seek romantic relationships exclusively with women. Her decision to attend the school is driven by her hope of finding a "pure maiden" to fall in love with. However, her plans are quickly disrupted upon meeting her roommate, Mariya Shidō, who appears to be the ideal girl Kanako has been searching for.

Mariya, however, harbors a significant secret: he is actually a cross-dressing boy posing as a girl to fulfill a family tradition tied to the school. Despite his true identity, Mariya maintains a flawless feminine appearance and demeanor, which constantly confounds Kanako. The series unfolds as a comedic exploration of Kanako's struggles to navigate her new environment, her growing frustration with Mariya's antics, and her attempts to reconcile her romantic aspirations with the reality of her situation.

The narrative is punctuated by a series of humorous and often absurd situations, many of which stem from Kanako's overactive imagination and her tendency to misinterpret events. Mariya frequently takes advantage of Kanako's naivety, orchestrating elaborate pranks that exacerbate her discomfort. Supporting characters, such as the stoic dorm supervisor Matsurika Shinōji and the eccentric school priest Father Kanae, add further layers of comedy and complexity to the story.

Throughout the series, Kanako's interactions with Mariya and the other students highlight themes of identity, deception, and the challenges of forming genuine connections. The dynamic between Kanako and Mariya serves as the core of the narrative, blending slapstick humor with moments of introspection. While the tone remains predominantly lighthearted, the series occasionally delves into more serious undertones, particularly in exploring Kanako's anxieties and Mariya's motivations for maintaining his disguise.

"Maria Holic" balances its comedic elements with a focus on character development, particularly in its portrayal of Kanako's growth as she confronts her fears and misconceptions. The series' episodic structure allows for a variety of comedic scenarios while gradually advancing the central relationship between Kanako and Mariya. The interplay between the characters and the school's unique setting creates a distinctive narrative that combines humor, romance, and a touch of absurdity.