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There is a significant demand for small, portable, and inexpensive analytical devices, which can be used in a wide range of sensing applications (e.g., food monitoring, detection of chemical, biological poisoning agents, environmental monitoring, medical diagnostics, military defense, etc.). Sensors based on organic semiconducting polymers, which are suitable for large-area, low-cost, flexible, and eventually single-use throwaway electronics, provide a unique opportunity in that sense. We report on low-operating voltage organic field-effect transistor devices, which can be used as sensors in electrolytes and liquid media, using a biofunctionalized, biocompatible, regioregular poly(3-hexylthiophene) semiconducting layer. Measurements in electrolytes and complex media relevant for cell analysis have shown that the devices can be directly used as ion-sensitive transducers and are suitable for in vitro biosensing applications. With the demonstration of biocompatible semiconducting polymeric devices, we have overcome a substantial hurdle for the realization of low-cost and mass-produced sensors, opening new possibilities of biological sensing using organic devices.