The　separation　of　glyceride　fish　oil　high　in　omega-3　polyunsaturated　fatty　acid　(ω-3　PUFA)　produced　by　multistage　enzymatic　transesterification　was　studied　by　multistep　molecular　distillation.　Firstly,　glycerides　and　by-products　in　the　transesterification　products　were　separated　by　primary　molecular　distillation.　When　the　distillation　pressure　was　5　Pa,　the　distillation　temperature　was　140　℃,　the　scraper　speed　was　240　r/min,　and　the　feeding　rate　was　0.2　mL/min,　the　yield　of　the　first-stage　heavy　phase　was　47.13%　and　the　content　of　ω-3　PUFA　in　it　was　53.76%.　Meanwhile,　high-temperature　gas　chromatography　(HTGC)　and　nuclear　magnetic　resonance　hydrogen　(1H-NMR)　spectroscopy　consistently　showed　that　the　heavy　phase　did　not　contain　fish　oil　ethyl　ester.　Secondly,　the　ω-3　PUFA　content　of　the　light　phase　increased　from　60.73%　to　78.01%　by　molecular　distillation　at　a　distillation　pressure　of　5　Pa,　a　distillation　temperature　of　110　℃,　a　scraper　speed　of　240　r/min,　and　a　condensation　temperature　of　20　℃.　After　conducting　secondary　enzymatic　transesterification　reaction　and　molecular　distillation　separation　on　the　enriched　first-stage　light　phase,　the　yield　of　the　second-stage　heavy　phase　was　78.01%　and　its　ω-3　PUFA　content　was　51.38%.　When　the　enrichment　step　was　omitted,　the　content　of　ω-3　PUFA　in　the　second-stage　heavy　phase　was　only　42.37%.　Multistep　molecular　distillation　combined　with　multistage　enzymatic　transesterification　can　be　used　to　prepare　glyceride-type　fish　oil　products　with　different　ω-3　PUFA　contents,　with　maximal　utilization　of　raw　materials　and　by-products.　©　2023　Chinese　Chamber　of　Commerce.　All　rights　reserved.