1,3-Dioleoyl-2-palmitoylglycerol (OPO) is an important component of triglycerides in human milk fat. The research focused on the optimization of different reaction factors to produce 1,3-dioleoyl-2-palmitoylglycerol as human milk fat substitute catalyzed by the immobilized lipase RMIM with tripalmitin (PPP) and oleic acid (OA) as substrates. The optimum reaction conditions were as follows: the molar ratio of PPP and oleic acid 1:6, the enzyme dosage 8% of the weight of substrates, the reaction temperature 50 ℃ and the reaction time 6 h. In the products, the concentrations of oleic acid and palmitic acid in sn-2 position were 54.86% and 87.64%, respectively, of the total fatty acids.