Methyllevulinate(ML)isapromisinggreencandidateforbio-baseddieselfuelandfueladditives.An efifcient enzyme-catalyzed process to synthesize ML from levulinic acid (LA) in methanol was developed. The catalytic activity of a series of lipases including Novozyme 435 (N435), NRTL IM, and 40086 was screened, and the N435 was identiifed as the optimal biocatalyst for the process. The effects of lipase amount, methyl tert-butyl ether (MTBE) volume, methanol to LA molar ratio, reaction temperature, and magnetic stirrer speed on LA conversion and ML yield were investigated. The response surface methodology was adopted to optimize the enzymatic conversion process, and the model validation experiments showed that the predicted values corresponded well with the experimental values. A LA conversion of 90.1％and a ML yield of 89.8％were achieved under reaction conditions covering:a temperature of 45 °C, a reaction time of 4.6 h, a N435 dosage of 26 mg, a methanol to LA molar ratio of 3.6:1, a MTBE volume of 3.85 mL, and a stirrer speed of 150 r/min. The N435 recycling experiment indicated that the lipase activity was quite high after 12 cycles. However, upon using crude LA prepared from carbohydrates as the reactant, the conversion of LA and the ML yield decreased due to impurities existing in the crude LA.