Abstract：In this study, we construct multilayer films of glucose oxidase/gold nanoparticles on the glassy carbon electrode (GCE) surface using 4-aminobenzoic acid (4-ABA), thionine (TH) and gold nanoparticles (Au NPs) as the covalent attachment cross-linkers. Electrochemical and analytical behavior of the enzyme electrodes was studied by cyclic voltammetry (CV) with hydroxyl ferrocene as mediator in the presence or absence of glucose. The results showed that the current value of the sensor vary with the glucose oxidase layers. In view of the long-term stability of the enzyme electrode and the structural simplicity, we have constructed two layers of glucose oxidase enzyme electrode. When the concentration of glucose solution reached 1×10－2 mol/L, the response current of the sensor would reach 2.47 μA and the response time is only 4.7 s. The bio-sensor linear range of detection is 3×10－5～1×10－3 mol/L, detection limit up to 5.8×10－6 mol/L. The sensor is simple and stable, and the results of the determination of glucose in human serum samples by the biosensor are close to that measured by hospital, so it has some value in use.