A copper oxide-overoxidized polypyrrole-reduced graphene oxide composite film(CuO-OPPy-ERGO/CCE) modified electrode was fabricated by using layer by layer electrochemical deposition technique.Firstly,a polypyrrole(PPy)-graphene oxide(GO) composite film was prepared by electropolymerization of pyrrole while using GO as the anionic dopant.After the electrochemical reduction in 0.10mol/L NaCl solution and peroxidation in 0.10mol/L NaOH solution, an overoxizied polypyrrole-reduced graphene oxide(OPPy-ERGO) composite film was obtained. Then, copper oxide was electrochemically deposited on this OPPy-ERGO film, and the modified electrode was obtained. The surface morphology and the electrochemical behavior of this modified electrode were investigated by scanning electron microscopy(SEM) and cyclic voltammetry. Furthermore, the electrochemical behavior of glucose on this electrode was also investigated carefully. The results showed that this modified electrode exhibited a high electrocatalytic activity and a good anti-interference ability for the oxidation of glucose. Under the optimum conditions, the linear range for the determination of glucose by amperommetry was in the range of 5.0×10-7-1.0×10-3mol/L.The detection limit(3Sb=3) and the sensitivity was 2.0×10-7 mol/L and 121.8μA/(mmol?L-1), respectively. The modified electrode was successfully used to determine glucose in human serum sample with recoveries of 96.0%-110.1%.