The invention relates to a preparation method of a human milk fat substitute. The method comprises the steps of: step 1) conducting an acid hydrolysis reaction on a raw material of high melting palm stearin subjected to chemical interesterification and alcohol leaching, and an acyl donor of rapeseed oil fatty acid by using Lipozyme RM IM as a catalyst, so as to reduce the total palmitic acid content and increase the relative content of site-two palmitic acid of the raw material; and step 2) deacidifying an intermediate product; and at the same time, calculating the amount of sunflower seed oil, rapeseed oil, palm kernel oil, algae oil and microbial oil added to the intermediate product according to the composition and distribution of fatty acids in human milk fat, by a grease adding model, so as to ensure composition and distribution of fatty acids in a final product meeting requirements of a human milk fat substitute, as well as realize a maximum addition amount. The invention adopts two steps to produce the human milk fat substitute, in order to reduce usage amount of free fatty acids in the acid hydrolysis reaction; meanwhile, two times of oil additions increase yield. Therefore, the invention greatly reduces the cost, and achieves a more than doubled yield compared with a similar products with a similar cost.