This paper proposed a two-step method to synthesize 1,3-Oleyl-2-Palmitoylglycerol (OPO) which is an important ingredient in the infant formula using an economical lipase from Candida sp. 99–125. In the first step, tripalmitin (PPP) is enzymatically alcoholyzed to monopalmitin (2-MP) by Candida sp. 99–125 lipase. MP is then recovered through solvent extraction at 78.03% yield and 98.01% purity, which are better than crystallization purification (75.85% yield and 86.37% purity). In the second step, the recovered 2-MP is solvent-free esterified with oleic acid, using the same lipase. OPO was obtained at a yield of 65%, including 75% palmitic acid in the Sn-2 position. This study has shown that the two-step method could give a higher yield of OPO than the previously reported one-step method using economical lipase.