Rhizopus oryzae lipase is one of the widely used industrial lipase. The enzymatic properties of recombinant rhizopus oryzae lipase were evaluated and it displayed strong 1,3 regiospecificity in hydrolyzing triglyceride; meanwhile, it showed 37.42% and 8.0% relative activity towards eicosapentaenoic acid(EPA) and docosahexenoic acid(DHA) compared with oleic acid in esterification reaction. Anchovy oil is one of the main sources of natural EPA/DHA and it contains 12.1% DHA. The result of 13C nuclear magnetic resonance spectrum showed that DHA in anchovy oil was preferentially located at sn-2 position of the glyceride. Based on above, recombinant rhizopus oryzae lipase was employed to hydrolyze anchovy oil to concentrate DHA, and the contents of DHA in produced 1,2-diacylglycerol and monoacylglycerol reached to 19.7% and 25.2% accompanying with a recovery yield of 65.6%. The results demonstrated that recombinant rhizopus oryzae lipase was a prospective lipase which could be used in enrichment of DHA by hydrolysis of anchovy oil.