The invention discloses a preparation method of a breast milk fat structural fat substitute, and belongs to the field of food additives and structural fat preparation. In the method, palm stearin is used as a raw material, the content of palmitic acid at the sn-2 position is improved through a random ester exchange reaction, and then the palm stearin and mixed fatty acid subjected to the random ester exchange reaction are catalyzed by sn-1,3 specific lipase to prepare a structural fat similar to breast milk fat, namely a breast milk fat structural fat substitute mainly comprising 1,3-dioleoicacid-2-palmitic acid triglyceride (OPO). An enzymatic reaction in the method provided by the invention adopts a solvent-free system, the repeated utilization rate of an enzyme preparation is high, andthe safety is good; therefore, the method provided by the invention has the advantages of stable source, low price, simple reaction process, short reaction time, high product content, 84.33% similarity of composition and components between an obtained product and the breast milk fat, controllable quality and the like in the production of the structural fat, and is suitable for large-scale industrial production.