A novel glucose biosensor based on immobilization of glucose oxidase in thin films of chitosan containing nanocomposites of graphene and gold nanoparticles (AuNPs) at a gold electrode was developed. The resulting graphene/AuNPs/chitosan composites film exhibited good electrocatalytical activity toward H(2)O(2) and O(2). The wide linear response to H(2)O(2) ranging from 0.2 to 4.2 mM (R=0.998) at -0.2V, high sensitivity of 99.5 microA mM(-1) cm(-2) and good reproducibility were obtained. The good electrocatalytical activity might be attributed to the synergistic effect of graphene and AuNPs. With glucose oxidase (GOD) as a model, the graphene/AuNPs/GOD/chitosan composite-modified electrode was constructed through a simple casting method. The resulting biosensor exhibited good amperometric response to glucose with linear range from 2 to 10 mM (R=0.999) at -0.2V and from 2 to 14 mM (R=0.999) at 0.5 V, good reproducibility and detection limit of 180 microM. Glucose concentration in human blood was studied preliminarily. From 2.5 to 7.5 mM, the cathodic peak currents of the biosensor decrease linearly with increasing the glucose concentrations. The graphene/AuNPs/GOD/chitosan composites film shows prominent electrochemical response to glucose, which makes a promising application for electrochemical detection of glucose.