A novel approach to fabricate an amperometric biosensor for the detection of glucose was described. By means of self-assembled technique and the opposite-charged adsorption, Nafion(Naf), Azure І (Azu), gold nanoparticles (nano-Au) and glucose oxidase (GOD) were immobilized on the gold electrode subsequently. The GOD immobilized on the nano-Au monolayer was stable and retained its functional activity. The stepwise self-assemble procedure of the immunosensor was further characterized by means of cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The factors influencing the performance of the resulting biosensor were studied in detail. The biosensor could detect the glucose in a range of 5.1×10-6~4.0×10-3mol/L.