In this study, the water level fluctuation zone of the Three Gorges Reservoir (TGR) was selected for investigation. A combination of chemical analysis and biological testing was utilized to investigate the effects of water fluctuation on the compounds and concentrations of estrogenic effector substances, as well as the estrogenic activity in soil (during the non-flooding period) and sediment (during the flooding period). The correlation between typical estrogens and estrogenic activity was analyzed. The study aimed to provide crucial data support for reservoir ecological safety and environmental health management. The distribution and concentration of 8 typical estrogens were analyzed using an ultra-performance liquid chromatography-mass spectrometry (UPLC-MS), while estrogenic activity was determined via the yeast estrogen screening assay (YES). Correlation analysis and concentration summation calculations were used to establish the relationships between chemical substances and biological effects. Among the 8 studied estrogens, only estrone (E₁) and ethinylestradiol (EE₂) were detected, with concentrations ranging from 0.025 ng/g to 2.667 ng/g. Sediments during the flooding period showed significantly higher estrogenic activity, with 17β-estradiol equivalents (EEQ) ranging from 0.637 ng/g to 6.987 ng/g. However, correlation analysis did not reveal a distinct correlation between the target compounds and estrogenic effects, as the detected chemicals only accounted for about 29.46% of the estrogenic activity. The results suggest that water fluctuation can influence the type and the concentration of estrogens in the water level fluctuation zone of the TGR, leading to increased estrogenic activity during the flooding period. The direct linkage between typical estrogens and estrogenic activity was not identified. Therefore, new methods and techniques are required to enhance identification accuracy and improve the management of the risk of estrogenic effects in the water level fluctuation zone.