Using internal standard Nandrolone-D3, a gas chromatography-mass spectrometric method (GC-MS) was developed for the identification and quantitative determination of testosterone propionate residues in the muscle tissues of various fishery products. Homogenized tissue samples were added with Nandrolone-D3 and saturated sodium chloride solution were extracted by Tert-butyl ether under ultrasonication. Clean-up was carried out with freezing, followed by Silica solid phase extraction (SPE). To enhance detection sensitivity, derivatization was achieved prior to GC-MS analysis with N-methyl-N-(trimethylsilyl)-trifluoroacetamide/iodotrimethylsilane/dithioeryth-ritol (MSTFA-TM IS-DTE). Quantitation using isotope-labeled internal standard was based on the ratio of peak area of testosterone propionate derivative to peak area of internal standard Nandrolone-D3 derivative in the selected ion monitoring (SIM) mode with electron impact (EI) source. The linearity ranged from 0.025 to 1.0 mg/L. The limit of quantitation (LOQ) was 1.0 μg/kg. At 1.0, 2.0, 10.0 μg/kg spiked levels, the mean recoveries were 74.1%-99.5%, and the relative standard deviations were 3.0%-8.3%. The application of this method was further demonstrated by analyzing various real samples from local markets.