A simple and effective glucose biosensor based on immobilization of glucose oxidase (GOD) in graphene (GR)/Nafion film was constructed. The results indicated that the immobilized GOD can maintain its native structure and bioactivity, and the GR/Nafion film provides a favorable microenvironment for GOD immobilization and promotes the direct electron transfer between the electrode substrate and the redox center of GOD. The electrode reaction of the immobilized GOD shows a reversible and surface-controlled process with the large electron transfer rate constant (ks) of 3.42±0.08 s−1. Based on the oxygen consumption during the oxidation process of glucose catalyzed by the immobilized GOD, the as-prepared GOD/GR/Nafion/GCE electrode exhibits a linear range from 0.5 to 14 mmol·L−1 with a detection limit of 0.03 mmol·L−1. Moreover, it displays a good reproducibility and long-term stability.