The invention relates to a preparation method of a human 
milk fat substitute, the human 
milk fat substitute prepared by the method, and use of the 
milk fat substitute. Specifically, edible fat is used as a 
raw material, and a random ester exchange reaction, a physical mixed reaction and a directed ester exchange reaction are performed, so that the human milk 
fat substitute is prepared. In the human milk 
fat substitute disclosed by the invention, compared with the total weight of 
fatty acid residues at an Sn-2 site of 
triglyceride of the human milk 
fat substitute, the content of 
palmitic acid residues at the Sn-2 site is 20wt%-60wt%, and the content of 
linoleic acid at the Sn-2 site is 3wt%-15wt%; compared with the total weight of the 
fatty acid residues in the 
triglyceride of the human milk fat substitute, the total content of the 
linolenic acid is 1wt%-5wt%. According to the method disclosed by the invention, the edible fat with broad sources is used as the 
raw material, and the technology is simple, so that the 
mass production of the human milk fat substitute of which the structure is similar to that of 
breast milk triglyceride in industry at a low cost can be realized. The human milk fat substitute prepared by the method disclosed by the invention is easy to absorb by babies, and promotion of the growth and the development of the babies is facilitated.