"Kono Naka ni Hitori, Imōto ga Iru!" centers around Shōgo Mikadono, a high school student who inherits his late father's vast fortune and business empire. However, there is a condition: to claim his inheritance, Shōgo must find a suitable bride before graduating high school. Complicating matters further, he learns that one of his potential brides is his long-lost younger sister, though her identity remains unknown. This revelation forces Shōgo to navigate his new life with caution, as he interacts with several female classmates who could potentially be his sister or his future wife.

The story unfolds at Mikadono Academy, where Shōgo transfers to begin his search. Among the key characters are Konoe Tsuruma, a childhood friend and classmate who harbors feelings for him; Miyabi Kannagi, a disciplined and serious student council president; Mana Tendō, a cheerful and energetic girl with a strong sense of justice; Rinka Kunitachi, a shy and reserved girl with a passion for art; and Mei Sagara, a mysterious and aloof transfer student. Each of these characters has a unique personality and background, and their interactions with Shōgo drive much of the narrative.

As Shōgo builds relationships with these girls, he must balance his personal feelings with the pressure of fulfilling his father's condition. The series explores themes of family, identity, and romance, with a focus on the mystery surrounding Shōgo's sister. The plot incorporates comedic and dramatic elements, often highlighting the misunderstandings and emotional conflicts that arise from Shōgo's predicament. The story progresses through a series of episodic encounters, gradually revealing clues about the identity of Shōgo's sister while developing the relationships between the characters. The narrative maintains a lighthearted tone, with occasional moments of tension and introspection, as Shōgo and the girls navigate their feelings and the challenges posed by their circumstances.