During the enzymatic production of biodiesel, methanol has a major inhibitory effect on enzyme activity whereas glycerol has a minor effect. Revitalization of the methanol-deactivated enzyme or pre-incubation of enzyme with various chemicals turned out to be unsuccessful. The stepwise feeding of methanol, a widely used conventional method for preventing methanol inhibition, was optimized in terms of the aliquot number and feeding interval to obtain a high conversion rate as well as a high degree of final biodiesel conversion. The use of six feedings of methanol with an equivalent molar ratio of 0.75 at 3-h intervals was found to be the optimal mode for preventing methanol inhibition; a biodiesel conversion rate of approximately 95% could be achieved within 20 h by using this method. Finally, to prevent contact between the undissolved methanol and the enzyme, methanol was pre-dissolved in water or biodiesel and fed to the mixture of soybean oil and the enzyme. This pre-dissolution method completely prevented enzyme inhibition, and a final biodiesel conversion rate of 82.3% was obtained.