Novozyme 435 was employed as biocatalyst for the transesterification of methyl laurate and glycerol in t-butanol producing glycerol monolaurate (GML). Emzymatic synthesis process was optimized by single-factor and orthogonal experiment using the content of glycerol monolaurate in the reaction system as the index. The results show that the optimum conditions were as follows: substrate molar ratio 1:5，substrate concentration 20%，reaction temperature 55 ℃，enzyme amount 7%，water content 20%，rotation rate 100 r/min，and reaction time 1 h. The content of glycerol monolaurate in the reaction system was 71.86% under the optimal synthesis conditions，After the recycle of unreacted methyl laurate，After purification the content of glycerol monolaurate in the final product was higher than 95%，with the maximum content 98.76%，while the content of glycerol dilaurate (GDL) was no more than 5%. Under the optimal conditions, the content of glycerol monolaurate decreased to 68.36% after being reused for six times, which was 71.75% in the first test.