Abstract: Abstract: The large consumption of fossil energy and the rising price of petroleum prompt us to look for an alternative resource for chemical production. Utilization of biomass resources can not only solve environmental problems, but also save resources and improve the economic value of biomass. Ethyl levulinate (EL) has a wide range of industrial applications. It can be used as an additive for various fuels and widely used in food, medicine, agriculture, rubber, manufacturing and other industries. At present, EL is mainly produced by chemical method. In contrast, the preparation of EL by biological method has the advantages of mild reaction conditions, low energy consumption and simple recovery of immobilized enzyme. It is considered as a new preparation method. In our previous studies, the preparation process of bio-levulinic acid has been reported. On this basis, lipase-catalyzed synthesis of EL from levulinic acid and ethanol was further studied. In this study, three lipases were screened, Nov435, Lipozyme TL IM and Lipozyme 40086, respectively. The results showed that Nov435 had good activity. Then, using NOV435 as biocatalyst, the effects of solvent volume (MTBE), enzyme amount, reaction time and molar ratio of ethanol to levulinic acid on EL yield were studied by single factor experiments. According to the experimental results, the reaction conditions were further optimized by response surface design. The experimental design includes 29 experiments and 5 repetitions at the central point to obtain the estimation of experimental errors. The quadratic equations of reaction temperature, lipase amount, MTBE volume and the molar ratio of ethanol to levulinic acid were obtained with EL yield as the optimized response. Using the equation, the optimum reaction conditions were obtained: reaction temperature 45 ℃, lipase amount 35.5 mg, MTBE 2.6 mL, molar ratio of ethanol to levulinic acid 1.7:1, rotating speed 150 r/min, reaction time 2.8 h. Under the optimum reaction conditions, the EL yield was estimated to be 87.6%. The validation experiment showed that the experimental result was 87.6%, which was very close to the predicted value. The catalyst reuse times of lipase under optimum conditions were studied and 12 repetitive experiments were carried out. The results showed that the recovery process of lipase was simple and easy to reuse. After five reuses, the high EL yield of 86.2% was still maintained. However, the EL yield decreased to 76.2% after 12 repeats due to the damage of lipase particles. In conclusion, this study showed that lipase-catalyzed EL production is an effective method, which provided an important basis for the preparation of EL by enzymatic method.