The tuna oil was refined by degumming， neutralization， bleaching and deodorization， and exploring the enrichment effect of low temperature crystallization method on triacylglycerols eicosapentaenoic acid(EPA) and docosahexaenoic acid (DHA) in refined fish oil. The results showed that after the treatment of refining process， all physicochemical indexes of fish oil reached the quality criterion of first grade refined fish oil； the content of saturated fatty acids decreased， but the contents of monounsaturated fatty acids and polyunsaturated fatty acids increased， EPA and DHA were 6.69% and 16.46%， respectively； the content of triacylglycerols in refined fish oil were 97.62%； the optimum conditions of enrichment were determined: the solvent was acetonitrile and acetone， the crystallization temperature was -50 ℃， the ratio of acetonitrile to acetone was 1 ∶ 12 (V/V)， the ratio of fish oil to compound solvent (acetonitrile and acetone) was 7 ∶ 40 (V/V)， the crystallization time was 150 min. Under these conditions， the contents of EPA and DHA were (12.83±0.34)% and (28.70±0.48)%， respectively， with corresponding yields of (40.10±1.07)% and (36.52±2.53)%.