The enzymatic production of biodiesel has been considered as an eco-friendly process. Candida antarctica lipase B (CalB) has been studied for its application for biodiesel production because of its high activity and stability. Enzyme deactivation caused by alcohol and effect of glycerol has to be resolved for the industrial application of this process. In traditional kinetic studies of biodiesel production, the effects of alcohol and oil were only considered in the kinetic equation, while the effect by glycerol was neglected. A new kinetic model incorporating glycerol effect is proposed in this paper. The proposed kinetic equation is applied by predicting the supplying rate of methanol in a fedbatch addition of methanol. The conversion rate was improved from 59.7% to 94.6% in a fed-batch by considering glycerol effect.