The dramatic progress in developing organic semiconductors with many virtues, including flexibility, high conductivity, solution processability, and biocompatibility, has aroused great research interest recently. Organic thin-film transistors (OTFTs), being an important type of organic semiconductor devices, have been successfully used in various high-performance sensors. This review will give a comprehensive description on the state-of-the-art chemical and biological OTFT-based sensors, including humidity, pH, ions, glucose, DNA, antibody-antigen, dopamine, bacteria, and cell-based sensors. Several essential aspects in this research field, including the properties of organic semiconductors, device physics, and the performance of different types of sensors, will be introduced in details.