Eicosapentaenoic acid (EPA) has been recognized as a promising nutrient to improve therapeutic efficacy for cancer patients. Nevertheless, there are certain limitations to the application of EPA due to its structural characteristics. To maximize the nutritive value of EPA, a type of medium- and long-chain triacylglycerol (MLCT) enriched with EPA was designed and synthesized using the lipase-catalyzed transesterification of medium-chain triglyceride (MCT) and EPA-enriched fish oil (FO). The optimum synthesis conditions for EPA-enriched MLCT used Lipozyme RM as catalyst, and had a substrate mass ratio (MCT/EPA-enriched FO) of 3:1, lipase loading of 80 g kg-1 , a reaction temperature of 60 °C, and a reaction time of 6 h. The MLCT content was as high as 80.79% after the transesterification reaction and the purification, and the content of MLCT containing EPA accounted for 70.21%. The distribution of EPA at the sn-2 position showed a significant increase in MLCT compared with the original substrate, from 18.89% to 26.93%. The in vitro digestion results demonstrated that MLCT had a significantly higher EPA bioaccessibility than the original substrate. Eicosapentaenoic acid-enriched MLCT was developed. This may provide a novel strategy for clinical nutritional intervention. © 2023 Society of Chemical Industry.