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Steroid hormones (SHs) are continuously released into the aquatic environment through various pathways after being excreted by humans and animals, interfere with the normal function of the endocrine system and may affect the physiology and reproduction of exposed aquatic life. To conduct a nationwide investigation of the occurrence and biological effects of SHs in surface river/steam water in China, we quantitated 27 selected SHs in 217 surface water samples by solid-phase extraction (SPE) tandem LC-MS/MS and used a recombinant yeast estrogen assay to screen extracts of the water samples for estrogenic activities. SHs were commonly found in the surface water samples, and their levels were typically in the ng L−1 range. Estrone (E1) and estriol (E3) were normally present in several to dozens of times higher concentrations than estradiol (E2) and 17-a-Ethinylestradiol (EE2). The high concentrations (mean > 1 μg L−1) of SumSHs were primarily obtained in areas under extreme water stress, specifically the eastern coastal areas. Source apportionment based on the profiles of the target compounds indicated that 54.5% of the SHs in target samples came from freshly discharged untreated sewage. The estrogen equivalent (EEQ(bio)) values ranged from 0.01 to 40.27 ng L−1, and the calculated EEQ (EEQ(cal)) values were generally lower than the corresponding EEQ(bio) values for all samples. E2 was the main contributor to the estrogenicity among the three estrogens, with a contribution ratio of 82.8%. The risk quotient values of E2 were highest and ranged from 1.55 to 782.95, and 76.0% of the target surface samples displayed the greatest environmental risk. We concluded that the impacts of SHs on humans in Chinese surface waters should not be ignored and that certain actions should be taken to decrease the levels of SHs in source waters, especially measures targeting SHs in untreated wastewater from the vast rural areas. Keywords: Steroid hormones, Estrogenic activities, Source apportionment, Environmental risk