"Senpai wa Otokonoko" centers on a high school setting, exploring themes of identity, self-discovery, and relationships. The protagonist, Makoto Hanaoka, is a transgender boy navigating the challenges of adolescence while concealing his gender identity from his peers. His life takes a turn when he befriends Aoi Saito, a cheerful and outgoing classmate who becomes intrigued by Makoto's reserved demeanor. As their friendship deepens, Aoi begins to suspect Makoto's secret, leading to moments of tension and emotional vulnerability.

The narrative delves into Makoto's internal struggles as he grapples with societal expectations, familial pressures, and his desire to live authentically. Aoi's unwavering support becomes a cornerstone of the story, highlighting the importance of acceptance and understanding. Secondary characters, such as Makoto's childhood friend Ryuji and his supportive teacher Ms. Takahashi, further enrich the narrative by providing additional perspectives on gender identity and the challenges faced by transgender individuals.

The plot progresses through a series of heartfelt and often poignant interactions, as Makoto gradually opens up about his identity to those around him. The series balances moments of lighthearted camaraderie with more serious discussions about discrimination and self-acceptance. By the end, "Senpai wa Otokonoko" offers a nuanced portrayal of the complexities of gender identity, emphasizing the significance of empathy and solidarity in overcoming societal barriers.