Abstract: Rates of each procedure composed of catalytic oxygen reduction reaction cycle with free multicopper oxidase as electro-catalyst in the presence of diffusional electron relay were determined by the spectroscopy,Clark-type oxygen electrode and cyclic voltammetry combined with rotating disc electrode technique,respectively. These rates were then compared to verify the rate-determining step in the catalytic reaction.Results from experiments indicate that the fastest step is the internal electron transfer in enzyme molecule(10³/s) followed by the enzymatic catalysis of oxygen transformation into water(91/s). The third procedure as for the rate was attributed to the electrochemical reduction reaction of the oxidation product of enzymatic catalytic reaction for electron mediator(0.19/s or 7.8 × 10^-3 cm/s) at the surface of base electrode. Mass transfer coefficients of the substrate,oxygen molecule,and oxidized/reduced electron mediator,2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium(ABTS),were evaluated to be 1.7 × 10^-3 cm/s,4.4 × 10^-4 cm/s and 6.3 × 10-4cm/s,respectively. Accordingly,chemical reaction rate in the enzyme catalytic electron mediator oxidation was determined to be 0.047/s. So it is apparent that the key procedure of the whole enzymatic catalysis cycle should be ascribed to the enzyme catalytic oxidation of electron mediator and the mass transfer of electron relay. The influence of parameters (including sorts of enzyme,specific activity of enzyme,enzyme concentration,types of mediators and their contents,temperature and pH value of electrolyte,etc.) on the enzyme electro-catalyzed oxygen reduction performance was investigated according to systematic modulation of those parameters. The results confirmed the above conclusions from the kinetic analysis.