Graphene was successfully prepared and well separated to individual sheets by introducing SO3−. XRD and TEM were employed to characterize the graphene. UV-visible absorption spectra indicated that glucose oxidase (GOx) could keep bioactivity well in the graphene-Au biocomposite. To construct a novel glucose biosensor, graphene, Au and GOx were co-immobilized in Nafion to further modify a glassy carbon electrode (GCE). Electrochemical measurements were carried out to investigate the catalytic performance of the proposed biosensor. Cyclic voltammograms (CV) showed the biosensor had a typical catalytic oxidation response to glucose. At the applied potential +0.4 V, the biosensor responded rapidly upon the addition of glucose and reached the steady state current in 5 s, with the present of hydroquinone. The linear range is from 15 μM to 5.8 mM, with a detection limit 5 μM (based on the S/N=3). The Michaelis-Menten constant was calculated to be 4.4 mM according to Lineweaver–Burk equation. In addition, the biosensor exhibits good reproducibility and long-term stability. Such impressive properties could be ascribed to the synergistic effect of graphene-Au integration and good biocompatibility of the hybrid material.