We report the synthesis of 1,3-dioleoyl-2-palmitoylglycerol (OPO) by a three-step method. Vinyl oleate was first synthesized by transvinylation between vinyl acetate and oleic acid. This was further reacted with glycerol at 35 °C for 8 h using 10% (w/v) Novozym 435 to synthesize 1,3-diolein. The 1,3-diolein content in the crude reaction mixture was 90.8% and was obtained at 82.3% (w/w) yield with 98.6% purity after purification. Finally, OPO was chemically synthesized by reacting purified 1,3-diolein with palmitic acid. 94.8% OPO was produced in the crude reaction mixture and the regiopurity of OPO after purification was 98.7% at 90.5% yield based on positional distribution analysis. This is an innovative approach for the synthesis of 1,3-diolein in a solvent-free system as an alternative to previously presented studies that applied solvent system.