The differences of 14 serum biochemical indexes and vitellogenin content between female and male migratory Anguilla japonica collected at the Yangtze River Estuary were compared, and the function of sex identification on female and male eels were established. The results showed that serum triglyceride (TG), low density lipoprotein (LDL-C), and calcium (Ca²⁺) level of the male eels were significantly higher than those of the female eels (P<0.05), while the albumin (ALB), and glucose (Glu) of the female eels were significantly higher than those of the male eels (P<0.05).There were no significant differences between the female and male eels on the levels of Alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (T-bil), total protein (TP), creatinine (CREA), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), and inorganic phosphorus (P) (P>0.05). Vitellogenin level of the female eels was significantly higher than that of the male eel (P<0.05). Base on the differences of the 14 serum biochemical indexes between female and male eels, two functions were established using stepwise discriminant analysis and entering independents together which could be used for sex determination of Anguilla japonica.