Abstract: PS was used as the raw material, palmitic acid as the acyl donors, and the nonspecific lipase Novozym 435 as catalyst to conduct the acidolysis to prepare Sn -2 position palmitic acid rich triglycerides, which could be raw material for subsequent human milk fat substitute preparation. Firstly, the ranges of all affecting factors were determined by single factor experiment and Box-Behnken center combination experimental design used to set up two times multiterm mathematical model, and then the response surface analysis was carried out. The result indicated that when the mass ratio of palmitic acid and PS was 1.09 ∶1, reaction time was 9.9h, reaction temperature was 54.7℃ and enzyme load was 21.9% , the Sn-2 palmitic acid of the obtained triglycerides could reach 60.59%, that could meet the requirements of human milk fat substitute preparation.