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In this study, we analyzed the eicosapentaenoic acid/docosahexaenoic acid (EPA/DHA) lipid composition of fish oil obtained through enzymatic treatment, fractional distillation and silica gel column purification, and further assessed EPA/DHA bioavailability. Lipid subclass composition information was obtained through ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS), and bioavailability tests were performed using the Caco-2 cell monolayer model. Results showed that enzymatic treatment improved the incorporation of EPA/DHA as diacylglycerol (DG) while silica gel column chromatography enriched the content of EPA/DHA as phosphatidylglycerol (PG) (12.58%) and phosphatidylethanolamine (PE) (4.99%). Furthermore, increasing the purity of EPA/DHA could improve its bioavailability and after 24 incubation, binding forms of triglyceride (TG) was superior to ethyl ester (EE) (p < 0.05) at the same purity level. Those findings are helpful to provide research basis for exploring the bioactivity of fish oil.