The invention discloses a preparation method of 1,3-dioleoyl-2-palmitoyl triglyceride, which comprises the following steps: carrying out esterification reaction on glycerol and palmitinic acid under the action of a catalyst to obtain tripalmitin; carrying out ester exchange reaction on the tripalmitin and oleic acid or oleate under the action of 1,3-specific lipase until the ester exchange rate reaches 30-60%, and removing free fatty acids or fatty acid esters in the reaction system; and adding oleic acid or oleate into the reaction system, continuing the reaction until the ester exchange rate reaches 60-90%, and separating and purifying the 1,3-dioleoyl-2-palmitoyl triglyceride from the reaction products. The method disclosed by the invention has the advantages of wide raw material sources, high economy, high safety, mild technological conditions, short reaction time, low energy consumption and low enzyme consumption; and the method can be used for preparing high-yield high-purity 1,3-dioleoyl-2-palmitoyl triglyceride, and can be used as a food nutrient reinforcer for blending infant formula milk powder.