An ultra performance liquid chromatography-tandem mass spectrometric（UPLC-MS/MS） method was developed for the simultaneous determination of steroid hormones in ovaries of zebrafish.Homogenized ovary samples were purified with NH2 and HLB solid phase extraction column after extracted with acetonitrile and ethyl acetate at 4 ℃.The samples were transported and eluted on an analytical column ACQUITY UPLC-BEH C18（2.1 mm×100 mm,1.7 μm）.All the analytes were detected in selected reaction monitoring（SRM） mode and quantified by the external matrix-matched standard method.The extraction efficiencies of different extraction solvents and cleanup effects of solid phase extraction columns for each analyte were further investigated.The average recoveries of 13 steroid hormones in spiked zebrafish ovary samples ranged from 74.8% to 127.0% with relative standard deviations（RSD） of 3.9%-15.8%.The results showed that the limits of detection（LODs,S/N=3） were in the range of 0.006-0.300 μg/kg.The method was sensitive and reliable,and could meet the requirements for the quantification of steroid hormones in zebrafish at physiological level.