Studies on estrogenic disrupting compounds (EDCs) occurrence and identification of main responsible compounds in river water discharged into the sea are of significance. In the present research, we screened estrogenic activities of 10 river water samples from 3 main rivers discharged into Bohai Sea in Tianjin using a recombinant two-hybrid yeast assay and chemical analysis by gas chromatography-mass spectrometry. All sample extracts induced significant estrogenic activity, with 17beta-estradiol equivalents (EEQ) of raw water ranging from 5.72 to 59.06 ng/L. Six most concerned EDCs in the river water samples including estrone, 17beta-estradiol, 17alpha-ethinylestradiol, estriol, diethylstilbestrol and estradiol valerate were determined, with their concentrations up to 50.70, 31.40, 24.40, 37.20, 2.56, and 8.47 ng/L, respectively. Through causality analysis by comparing the EEQ values of yeast assay and chemical analysis, 17alpha-ethinylestradiol and 17beta-estradiol were identified as the main contributors to the estrogenic effects of the river samples, accounting for the whole estrogenic activities (62.99% to 185.66%), and estrogen antagonistic compounds might presented in the heavy polluted water samples. The proposed approach using both chemical analysis and bioassay could be used for identification and evaluation of the estrogenic activity of EDCs in river water.