Organic thin film transistors (OTFTs) enable rapid and label‐free high‐sensitivity detection of target analytes due to their low cost, large‐area processing, biocompatibility, and inherent signal amplification. At the same time, the freedom of synthesis, tailorability, and functionalization of organic semiconductor materials and their ability to be combined with flexible substrates make them one of the ideal platforms for biosensing. However, OTFTs‐based biosensors still face significant challenges, such as unexpected surface adsorption, disordered conformation, inhomogeneous graft density, and flexibility of probe molecules that biological sensing probes would face during immobilization. In this review, efficient immobilization strategies based on OTFTs biological sensing probes developed in the last 5 years are highlighted. First, the biosensors are classified according to their sensing interface. Second, a comprehensive discussion of the types of biological sensing probes is presented. Third, three commonly used methods for immobilizing biological sensing probes and their challenges are briefly described. Finally, the applications of OTFTs‐based biosensors for liquid phase detection are summarized. This review provides a comprehensive and timely review of optimization in sensing interface engineering so that efficient immobilization of biological sensing probes with sensing interfaces will contribute to the development of high‐performance OTFTs‐based biosensors.