This study investigated estrogenic activity of water, sediment, and fish bile of the Pearl River in southern China by effect-directed analysis based on in vitro yeast screen assay and chemical analysis. Results showed higher estradiol equivalents (EEQ) for surface water in dry season than in wet season. Simple risk assessment suggested that high estrogenic risk would be expected in Shima River and Danshui River receiving discharge of effluents from cities in the region. Fractionation and effect-directed analysis showed that estrogenic activity mainly occurred in relatively polar fractions of surface water. Seven target estrogenic compounds (bisphenol A, 4-nonylphenol, 4-tert-octylphenol, 17α-ethynyl estradiol, estrone, diethylstilbestrol, and 17β-estradiol) only accounted for part of the measured estrogenic activity, with the rest contributions from other potential estrogenic chemicals such as phenols. Findings from this study suggest that fish in the river could be affected by those estrogenic chemicals. Proper measures should be taken to reduce the estrogenic activity in wastewaters before they are discharged into the riverine system in order to protect aquatic organisms.