Thin film glucose biosensors were fabricated with organic/inorganic hybrid films based on glucose oxidase (GOx) and Prussian Blue nano-clusters. The biosensors composed of hybrid films were characterized by the low operating potential and the advantage to interference-free detection. In this research, we employed two kinds of thin films for GOx immobilization: Langmuir-Blodgett (LB) and self-assembled monolayer (SAM). The LB film immobilizes GOx in its inside through the electrostatic force, while the SAM immobilizes GOx with the covalent bond. The sensors with LB film produced a relatively high current signal, while the non-linear behavior and a low stability were recognized. On the other hand, the sensors with SAM presented a good linear relationship and a very stable performance.