The working principle of enzyme-based biofuel cells (EBFCs) is the same as that of conventional fuel cells. In an EBFC system, the electricity-production process is very intricate. Analysis requires a mathematical model that can adequately describe the EBFC and predict its performance. This paper develops a dynamic model simulating the discharge performance of the anode for which supported glucose oxidase and mediator immobilize in the EBFC. The dynamic transport behavior of substrate, redox state (ROS) of enzyme, enzyme-substrate complex, and the mediator creates different potential changes inside the anode. The potential-step method illustrates the dynamic phenomena of substrate diffusion, ROS of enzyme, production of enzyme-substrate complex, and reduction of the mediator with different potential changes.