Abstract: A method of liquid chromatography-tandem mass spectrometry（LC-MS/MS） was developed for the simultaneous determination of estrogen, progestogen and androgen in aquatic products muscle tissues. The sample were extracted by tert-butyl methyl ether and ethyl acetate, purified on C18 solid phase extraction column, eluted by methanol. After XBridge RP-18 column separation using 0.02% ammonia-methanol as mobile phase, the eluents were qualitatively and quantitatively determined under multi-reaction monitoring scan type with tandem mass analyzer using positive polarity mode for the progestogen, androgen and estradiol benzoate, negative polarity for the estrogen respectively, the quantification was performed with internal standard method. The limit of detection is 0.5—2 μg/kg（S/N≥3）. Spike recoveries are 73.0%—120% and 79.0%—108% in 2 μg/kg and 10 μg/kg with relative standard deviation of 5.3%—15% and 2.3%—8.2%. The method is effective, simple and sensitive, and suitable for the determination and confirmation of 27 kinds of sex hormones in aquatic products.