Enzymatic biofuel cells with the possible use of nontoxic fuels under moderate operating conditions have the potential to power portable devices and medical assistance devices used near the body. Moving toward the rational design of an enzyme electrode with a high current density, the present study clarified the factor that limits the current density of enzymatic biofuel cells. Comparison of the experimental results of a thin-film electrode with a physically adsorbed mediator and enzyme with the model calculation using parameters obtained from the enzyme adsorption and electrochemical experiments showed that the ratio between the Michaelis constants for the mediator and the effective concentration of the mediator was the important parameter. The model calculation was then used to design the electrode of biofuel cells with a high current density, which revealed the importance of the immobilization method of the mediator.