Triacylglycerols (TAG) are differences in fatty acid distributions between infant formula and human milk. In this study, fish oil (Tilapia, Golden pompano, Tiger grouper, and Basa) showed the potential as the source of human milk fat substitutes by comparing TAG profiles with infant formula and human milk. The total lipids and TAG of fish were concentrated in the by-products of fish (head and viscera) and contained high levels of palmitic acid, oleic acid, and linoleic acid. Compared with infant formula, fish oil was closer to human milk in sn-2 fatty acid distribution, and sn-2 palmitic acid level in fish oil exceeded 52 % of total palmitic acid, Golden pompano head was the highest (64.46 %). Further research showed that the content of sn-2 palmitoyl TAG (OPO and OPL dominated) increased from 157.16 mg/g TAG to 305.18 mg/g TAG by isopropanol enrichment (solid–liquid ratio: 1:4, temperature: −12 °C, time: 4 h).