In the past decades, a variety of organic field‐effect transistor (OFET) based sensors have been reported, holding great promise in physical sensing, chemical sensing, biosensing, medical sensing and functionalized integrated sensor arrays, thanks to the superiorities of OFETs, including fabrication versatility and high sensitivity to external stimulus. However, the sensing performance still needs to be enhanced to meet practical application requirements, especially high sensitivity and short response time. Among all the effective strategies, ultrathin organic semiconductor (OSC) film devices show significantly promoted sensing performance, and the fabrication methods to obtain these ultrathin OSC films are of importance and diversity. Here, a brief review focused on ultrathin (including molecular monolayer) OSC film fabrication methods is presented, including high‐vacuum thermal evaporation, solution‐processable methods, and the approach of combining ultrathin films with heterojunctions. Additionally, applications of ultrathin OFET‐based chemical and biological sensors are summarized. Finally, the outlook and challenges ahead are discussed. We hope this review can offer guidance for the fabrication methods and potential applications of ultrathin OFET‐based chemical and biological sensors in the future. © 2020 Society of Industrial Chemistry