Abstract: Great potential harm from livestock manure is commonly found in estrone (E1), 17α-E2(17α-estradiol), 17β-E2(17β-estradiol) and E3(estriol).Gas chromatography/mass spectrometry (GC-MS) method was developed for the analysis and quantification of the four typical steroid estrogens in pig manure samples.The pre-treatment method was carefully optimized,including the options for the ultrasonic extraction solvent,extraction time,solid phase extraction eluent,derivatization temperature and time.The results showed that the detection limits for E1,17α-E2,17β-E2 and E3 in pig manure were 8.0,2.5,5.1 and 3.3 μg/kg under the optimal experimental conditions.The average spiked recoveries were in the range of 92.3% to 137.4%.