A method of high performance liquid chromatography combined with gel permeation chromatography（GPC/HPLC) was developed for the simultaneous analysis of methyltestosterone（MET)and diethylstilbestrol（DES) in aquatic products.The homogenized samples were extracted with diethyl ether,then cleaned by GPC using ethyl acetate-cyclohexane（1∶1)as mobile phase,follwed by a further HLB solid phase extraction.The targeted compounds were detected by HPLC-ultraviolet detector at the wavelength of 254 nm,using methanol-water（72∶28) as mobile phase,and quantified by the external standard method.The calibration curves of two compounds were linear in the concentration range of 0.02-2.0 mg/L with correlation coefficients more than 0.999.The average recoveries of MET and DES at spiked concentrations of 10.0,50.0,200 μg/kg ranged from 84% to 93%,with relative standard deviations（RSDs) less than 6.0%.The LOQs of MET and DES were 6.0 μg/kg and 5.0 μg/kg,respectively.