Estrogenic and dioxin activities in river water and effluents of sewage treatment plants were evaluated by different in vitro assays. The expression of β-galatosidase in yeast cells (Yeast two-hybrid assay) was used as an indicator of pollutants effects. Estrogenic activities were estimated using medaka estrogen receptor (mER) and human estrogen receptor (hER) yeast while the dioxin activities were measured by Aryl hydrocarbon receptor (AhR) yeast. Five samples were collected including two river water (RW) samples and three effluents (MW) samples. Target chemicals were extracted using a series of solvents which are hexane/dichloromethane, acetone/dichloromethane and methanol. Standard dose-response curves were established with 3 estrogenic chemicals (estradiol, nonylphenol and bisphenol A) and 3 dioxin-like chemicals (naphthoflavone, comestrol and equol). The results showed that both estrogen receptors (i.e., mER and hER) were suitable to be applied into the assessment of micropollutants` estrogenicity, in which, the sensitivity of mER to estrogenic activity was higher than that of hER. AhR also showed a suitable sensitivity to target dioxins. Estrogenic activity of MW-1 was highest among the samples in both mER and hER yeast assays. The estrogenic activities of two other effluents samples, however, were lower than that of MW-1 and other RW samples. This indicates that river water may receive estrogenic-polluted water other than the effluents of STPs. Similarly, dioxin activities in RW samples were higher than that of effluents. It is suggested that the untreated agriculture wastewater could be the reason. This study contributes to the establishment and application of risk assessment of micropollutant in the environment.