Polypyrrole film (PPy) was prepared by cyclic voltammetry on the surface of glassy carbon electrode and three-dimensional graphene (GN) was prepared by hydrothermal reduction method in this study. A novel enzyme-based electrode was explored for the oxidization of glycerol through using glycerol kinase (GK) and glycerol triphosphate oxidase (GPO) as catalysts, three-dimensional graphene (GN) as the carrier, polypyrrole (PPy) as the mediator, and the Nafion solution as the binder. The glycerol enzyme-based electrode could provide good electrical transfer among the enzyme, mediator and the electrode surface. In this paper, the polymerization condition of pyrrole was investigated by scanning electron microscope and electrochemical methods. The modified materials and working conditions of the enzyme electrode were optimized and their properties were evaluated by electrochemical method. The results showed that the performance of modified electrode was optimal when polymerization cycle was 8, and the enzyme-based electrode conducted a good catalytic performance with a strong response for glycerol oxidization in 0.2 mmol/L phosphate buffer solution (PBS) at pH 7.0,. The peak catalytic current reached 46.2 μA and the current density reached 677.6 μA/cm2.