[Objectives] In the current study, the water level fluctuation zone of the Three Gorges Reservoir (TGR) was selected, in which chemical analysis was combined with biological testing to investigate the effects of water fluctuation on the compounds and concentrations of estrogenic effector substances and the estrogenic activity in soil (non-flooding period) and sediment (flooding period). The correlation between typical estrogens and estrogenic activity was analyzed. The study hence could provide important data support for reservoir ecological safety and environmental health management. [Methods] The distribution and concentration of 8 typical estrogens were analyzed by an ultra-performance liquid chromatography-mass spectrometry (UPLC-MS), and the estrogenic activity was determined by a Yeast Estrogen Screening assay (YES). Correlation analysis and concentration summation calculations were used to establish the correlations between chemical substances and biological effects. [Results] Only estrone (E1) and ethinylestradiol (EE2) were detected with the ranges of 0.02-2.67 ng/g from the 8 studied estrogens. The sediments of the flooding period showed significantly estrogenic activity, which the 17β-estradiol equivalents (EEQ) ranging from 0.637-6.987 ng EEQ/g. Correlation analysis did not show distinctly correlation between the target compounds and estrogenic effects, which the detected chemicals only contributed about 29.46% of the estrogenic activity. [Conclusions] Water fluctuation could affect the type and concentration of estrogens in the water level fluctuation zone of the TGR, with the estrogenic activity increased during flooding period. The linkage between typical estrogens and estrogenic activity was not found. Therefore, new methods and techniques are required in order to increase the identification accuracy and improve the management on the risk of estrogenic effects in the water level fluctuation zone.