The early determination of disease-related biomarkers can significantly improve the survival rate of patients. Thus, a series of explorations for new diagnosis technologies, such as optical and electrochemical methods, have been devoted to life and health monitoring. Organic thin-film transistor (OTFT), as a state-of-the-art nano-sensing technology, has attracted significant attention from construction to application owing to the merits of being label-free, low-cost, facial, and rapid detection with multi-parameter responses. Nevertheless, interference from non-specific adsorption is inevitable in complex biological samples such as body liquid and exhaled gas, so the reliability and accuracy of the biosensor need to be further improved while ensuring sensitivity, selectivity, and stability. Herein, we overviewed the composition, mechanism, and construction strategies of OTFTs for the practical determination of disease-related biomarkers in both body fluids and exhaled gas. The results show that the realization of bio-inspired applications will come true with the rapid development of high-effective OTFTs and related devices.