Lung cancer is the most common cause of cancer-related death in humans. Therefore, development of sensitive, selective and non-destructive diagnose method of lung cancer is of paramount importance. In this work, an electron-poor fluorophore, perylene bisimide (PBI), was modified with cholesterol (Chol) at its two bay positions, resulting in a derivative of PEBC with improved solubility in organic solvents and enhanced self-assembly properties. Photophysical and SEM studies revealed that the compound as developed has a great tendency to form aggregates in different solvents. Accordingly, a fluorescent film with optimized fibrillar networked structures was fabricated by adjusting the assembly behavior of PEBC in mixed solvents. Sensing performance studies demonstrated that the as prepared film is super-sensitive and selective to the presence of aniline and o-toluidine in air. Based on the studies, an aniline, an important biomarker of lung cancer, sensor was constructed. Sensor’s performance studies demonstrated that aniline and its relevant o-toluidine, another biomarker of lung cancer, could be sensitively, selectively, reversibly and fast detected. The response time is less than 1s, the calculated detection limit (DL) is ∼15ppb, and presence of common organic amines and solvents show little effect upon the detection. Moreover, the sensor works well when simulated exhale from lung cancer patients is employed as a sample, indicating its great potential in the diagnosis of lung cancer.