An organic thin film transistor (OTFT), a method of manufacturing the same, and a biosensor using the OTFT are provided. The OTFT includes a gate electrode, a gate insulating layer, source and drain electrodes, and an organic semiconductor layer disposed on a substrate and further includes an interface layer formed between the gate insulating layer and the organic semiconductor layer by a sol-gel process. The gate insulating layer is formed of an organic polymer, and the interface layer is formed of an inorganic material. The OTFT employs the interface layer interposed between the gate insulating layer and the organic semiconductor layer so that the gate insulating layer can be protected from the exterior and adhesion of the gate insulating layer with the organic semiconductor layer can be improved, thereby increasing driving stability. Also, since the OTFT can use a plastic substrate, the manufacture of the OTFT is inexpensive so that the OTFT can be used as a disposable biosensor.