•Graphene/nano-Au composite was synthesized by electrochemical co-reduction method in one step.•Glucose oxidase achieves direct electrochemistry on the graphene/nano-Au composite film.•The glucose biosensor shows a high sensitivity of 56.93μAmM−1cm−2 toward glucose.•Glucose was detected with a wide linear range and low detection limit. A simple, green and controllable approach was employed for electrochemical synthesize of the graphene/nano-Au composites. The process was that graphene oxide and HAuCl4 was electrochemically co-reduced onto the glassy carbon electrode (GCE) by cyclic voltammetry in one step. The obtained graphene/nano-Au/GCE exhibited high electrocatalytic activity toward H2O2, which resulted in a remarkable decrease in the overpotential of H2O2 electrochemical oxidation compared with bare GCE. Such electrocatalytic behavior of the graphene/nano-Au/GCE permitted effective low-potential amperometric biosensing of glucose via the incorporation of glucose oxidase (GOD) with graphene/nano-Au. An obvious advantage of this enzyme electrode (graphene/nano-Au/GOD/GCE) was that the graphene/nano-Au nanocomposites provided a favorable microenvironment for GOD and facilitated the electron transfer between the active center of GOD and electrode. The immobilized GOD showed a direct, reversible redox reaction. Furthermore, the graphene/nano-Au/GOD/GCE was used as a glucose biosensor, displaying a low detection limit of 17μM (S/N=3), a high sensitivity of 56.93μAmM−1cm−2, acceptable reproducibility, very good stability, selectivity and anti-interference ability.