A method for preparing an eicosapentaenoic acid ethyl ester and a docosahexaenoic acid ethyl ester from a raw material fat comprising eicosapentaenoic acid and docosahexaenoic acid comprising the steps of: (a)treating the raw material fat with a lipolytic enzyme under a condition of ethyl-esterifying a fatty acid; (b) fractionating the treated substance of the step (a) into a glyceride fraction and an ethyl-esterified free fatty acid fraction; (c) ethyl-esterifying the glyceride fraction obtained in the step (b); (d) purifying and fractionating the ethyl-esterified glyceride fraction obtained in the step (c) into: a fraction comprising more docosahexaenoic acid ethyl ester than the ethyl-esterified substance of the glyceride fraction in the step (c) and comprising less eicosapentaenoic acid ethyl ester than the ethyl-esterified substance of the glyceride fraction in the step (c); and a fraction comprising more eicosapentaenoic acid ethyl ester than the ethyl-esterified substance of the glyceride fraction in the step (c) and comprising less docosahexaenoic acid ethyl ester than the ethyl-esterified substance of the glyceride fraction in the step (c); (e) purifying and fractionating the ethyl-esterified free fatty acid fraction obtained in the step (b) into: a fraction comprising more docosahexaenoic acid ethyl ester than the ethyl-esterified free fatty acid fraction in the step (b) and comprising less eicosapentaenoic acid ethyl ester than the ethyl-esterified free fatty acid fraction in the step (b); and a fraction comprising more eicosapentaenoic acid ethyl ester than the ethyl-esterified free fatty acid fraction in the step (b) and comprising less docosahexaenoic acid ethyl ester than the ethyl-esterified free fatty acid fraction in the step (b); (f) mixing the fraction of in the step (d) with the fraction of in the step (e); (g) mixing the fraction of in the step (d) with the fraction of in the step (e); (h)further purifying the mixture in the step (f) to obtain a purified substance comprising the docosahexaenoic acid ethyl ester; and (i) further purifying the mixture in the step (g) to obtain a purified substance comprising the eicosapentaenoic acid ethyl ester. (a)treating the raw material fat with a lipolytic enzyme under a condition of ethyl-esterifying a fatty acid; (b) fractionating the treated substance of the step (a) into a glyceride fraction and an ethyl-esterified free fatty acid fraction; (c) ethyl-esterifying the glyceride fraction obtained in the step (b); (d) purifying and fractionating the ethyl-esterified glyceride fraction obtained in the step (c) into: a fraction comprising more docosahexaenoic acid ethyl ester than the ethyl-esterified substance of the glyceride fraction in the step (c) and comprising less eicosapentaenoic acid ethyl ester than the ethyl-esterified substance of the glyceride fraction in the step (c); and a fraction comprising more eicosapentaenoic acid ethyl ester than the ethyl-esterified substance of the glyceride fraction in the step (c) and comprising less docosahexaenoic acid ethyl ester than the ethyl-esterified substance of the glyceride fraction in the step (c); a fraction comprising more docosahexaenoic acid ethyl ester than the ethyl-esterified substance of the glyceride fraction in the step (c) and comprising less eicosapentaenoic acid ethyl ester than the ethyl-esterified substance of the glyceride fraction in the step (c); and a fraction comprising more eicosapentaenoic acid ethyl ester than the ethyl-esterified substance of the glyceride fraction in the step (c) and comprising less docosahexaenoic acid ethyl ester than the ethyl-esterified substance of the glyceride fraction in the step (c); (e) purifying and fractionating the ethyl-esterified free fatty acid fraction obtained in the step (b) into: a fraction comprising more docosahexaenoic acid ethyl ester than the ethyl-esterified free fatty acid fraction in the step (b) and comprising less eicosapentaenoic acid ethyl ester than the ethyl-esterified free fatty acid fraction in the step (b); and a fraction comprising more eicosapentaenoic acid ethyl ester than the ethyl-esterified free fatty acid fraction in the step (b) and comprising less docosahexaenoic acid ethyl ester than the ethyl-esterified free fatty acid fraction in the step (b); a fraction comprising more docosahexaenoic acid ethyl ester than the ethyl-esterified free fatty acid fraction in the step (b) and comprising less eicosapentaenoic acid ethyl ester than the ethyl-esterified free fatty acid fraction in the step (b); and a fraction comprising more eicosapentaenoic acid ethyl ester than the ethyl-esterified free fatty acid fraction in the step (b) and comprising less docosahexaenoic acid ethyl ester than the ethyl-esterified free fatty acid fraction in the step (b); (f) mixing the fraction of in the step (d) with the fraction of in the step (e); (g) mixing the fraction of in the step (d) with the fraction of in the step (e); (h)further purifying the mixture in the step (f) to obtain a purified substance comprising the docosahexaenoic acid ethyl ester; and (i) further purifying the mixture in the step (g) to obtain a purified substance comprising the eicosapentaenoic acid ethyl ester.