When Kanako Miyamae transferred to a new all-girls school, she hoped to find true love. What she found instead was the beautiful Mariya Shidou, a charming classmate Kanako met on her first day. But Mariya has one flaw: she is actually a cross-dressing boy! After discovering Mariya's secret and becoming stuck with him as her roommate, Kanako's life has become a series of misfortunes. All she wants is to find the girl of her dreams, which is hard to do with the meddling of the sadistic Mariya and his sharp-tongued maid Matsurika Shinouji.

Maria†Holic Alive continues the story of Kanako as she deals with being Mariya's roommate and aims to find true love, while enduring high school itself.