A simple, rapid and sensitive method was developed for determination of 17α-methyltestosterone in aquatic products by extraction with subcritical 1,1,1,2-tetrafluoroethane (R134a) extraction and high performance liquid chromatography (HPLC). Response surface methodology (RSM) was adopted to optimise extraction pressure, temperature and co-solvent volume. The optimum extraction conditions predicted within the experimental ranges were as follows: pressure 5MPa, temperature 31°C, and co-solvent volume 3.35ml. The analysis was carried out on XDB-C18 column (4.6mm×250mm, 5μm) with the mobile phase acetonitrile–water (55:45, v/v), flow rate 0.8ml/min, temperature 30°C and wavelength 245nm. Good linearity of detection was obtained for 17α-methyltestosterone between concentrations of 50–250ng/ml, r2=0.999. The method was validated using samples fortified with 17α-methyltestosterone at levels of 10, 30 and 50ng/g, the mean recovery exceeds 90%, and the RSD values were less than 10%.