A stable, ultrasensitive, and fully reversible fluorescent sensing film for organic amines has been fabricated by assembling cholesterol (Chol)-derived perylene bisimide on a glass plate surface. The compound exhibits excellent film formation properties and forms well-defined nanofibers, as evidenced by SEM and AFM measurements. It has been revealed that besides the molecular structure of the specially designed perylene derivative, the existence of nanofibers in the film is another key factor to endow the film with superior sensing ability for organic amines, including aniline. The detection limit of the amine is ca. 150.0 ppt in the vapor phase and at room temperature. Furthermore, the sensing process is free of interference from common organic solvents, nitroaromatics, and particularly phenols, which makes the film a potential candidate to be used in lung cancer diagnoses and related applications.