The purpose of this paper was to investigate the fatty acid release characteristics of OPO structural lipids in simulating the gastrointestinal digestive environment of infants. According to the characteristics of fatty acid composition of breast milk， OPO structural lipids （O Group） were prepared in this study， and mixed vegetable oil （Z group） in infant formula was used for comparison. The fatty acid composition and the distribution of fatty acids at the Sn-2 position were measured. The changes in fatty acid release rate， particle size and zeta potential during digestion were analyzed， and the composition of free fatty acids and monoglycerides in the digested products was determined. The results showed that under the same treatment conditions， the particle size of the O group was concentrated at 198 nm， and the Z group was mostly distributed at 229 nm. The difference in particle size distribution reflected the significant difference in fatty acid release rate during the initial digestion stage （P<0.05）. After 30 min of the simulated gastric fluid， the fatty acid release rate of the O group was 12.83%， and the Z group was 8.92%. After the in vitro gastrointestinal digestion， the final release rate of fatty acids in O group was 62.67%， while the final release rate of fatty acids in Z group was only 51.83%. The analysis concluded that there was a difference in the distribution of Sn-2 fatty acids in the O group and Z group （P<0.05）. Thin-layer chromatography confirmed that the content of Sn-2 palmitic acid in the O group was higher and closer to that of breast milk， and showed higher fatty acid release rate when simulated in vitro digestion. After methylation of the digested product， GC-MS analysis showed that the content of free unsaturated fatty acid and palmitic acid monoglyceride in the O group was higher than that in the Z group. Therefore， comparing the digestion of OPO oils and mixed vegetable oils in vitro， it lays a theoretical foundation for the development of formulas that are more conducive to digestion and absorption of infants.