Enzymatic biodiesel production kinetics under previously optimized conditions were investigated. Waste frying oil (WFO) was used as the raw material, Novozym 435 as catalyst, methanol as acyl acceptor and tert-butanol as co-solvent. To investigate pure transesterification kinetics improving product properties, 3Å molecular sieves were incorporated into the reaction to provide an anhydrous medium avoiding the side reactions of hydrolysis and esterification. The effects of either WFO or methanol on the reaction rate were analyzed separately. The reaction was described by a Ping Pong mechanism and competitive inhibition by methanol. The results obtained in the kinetics study were applied in the operation of a semi-continuous reactor for biodiesel production. The operational conditions of each reaction cycle were: methanol-to-oil ratio 8/1 (mol/mol), 15% (wt) Novozym 435, 0.75% (v/v) of tert-butanol, 44.5°C, 200 rpm and 4h of reaction time. The enzymes were successively reused by remaining in the reactor during all the cycles. Under these conditions, biodiesel production yields higher than 80% over 7 reaction cycles were observed. Both the kinetics study and the reactor operation showed that Novozym 435 was not inhibited at high methanol concentrations and that the kinetics of the proposed enzymatic process could be comparable to the conventional chemical process.