An electrochemical oxidation process has been developed to fabricate enzyme electrodes, immobilized with an enzyme glucose oxidase (GOx) and a polymeric mediator poly(vinylferrocene) (PVFc). The ferrocene moiety of PVFc transfers electrons successfully between the GOx and the electrode surface, and the macromolecular nature of PVFc helps to minimize the loss of mediator molecules. Enzyme electrodes fabricated by this process respond rapidly to glucose, with steady state currents being achieved in less than 10 s. Current responses to glucose concentrations were investigated under different operating conditions, such as pH, temperature, oxygen and anion interference. A mathematical model describing the steady state amperometric operation of the enzyme electrode has been derived. The reaction steps include diffusion, heterogeneous enzymatic catalysis, electron mediation and electrochemical regeneration of mediators. Simplified equations, corresponding to certain kinetic characteristics, are presented. Each case shows its unique relationship between the inverse current and the inverse analyte concentration. Therefore, a diagnostic plot of inverse current vs. inverse analyte concentration allows rate-limiting step(s) to be identified immediately. Experimental data obtained from known systems confirm the validity of this model.