Abstract: In order to solve trace-level steroid estrogen pollution in groundwater and surface water, a SPE-GC-MS approach to determine five steroid estrogens (SEs), E1, 17α-E2, 17β-E2, EE2 and E3, by optimizing of solid phase extraction (SPE), derivatization conditions and the secondary purification process of complex samples has been developed. The results show that Oasis HLB column, ethyl acetate elution and derivatizing at 40℃ for 20 min can achieve the best results for extraction. Moreover, the Generik NAX column activated by methyl is suitable for the secondary purification of complex samples. The linear ranges of E1, 17α-E2, and 17β-E2 are 5-1000 ng/L, whereas those of EE2 and E3 are 10-1000 ng/L. The detection and quantitation limits are 2-3 ng/L and 6.5-10 ng/L, respectively. The standard solution added recoveries of water samples range from 80% to 120%. The relative standard deviations of daily peak areas in the SEs determination range from 6.8% to 10%. This method was used to determine the SEs pollution levels of waters from pond, river, groundwater and sewage treatment plant effluent and results show that this detection technique can be effectively applied to the identification and evaluation of estrogen risk in surface water and groundwater samples.