Abstract: The structural integrity of aquatic ecosystems is the core of aquatic ecological protection and restoration, with its systematic assessment serving as a pivotal determinant for achieving sustainable watershed management. During the evaluation process, factors such as the structural composition of biological communities, habitat conditions, and food web structure should be fully considered. This study takes the main stem of the Three Gorges Reservoir as an example, and by reviewing existing domestic and international aquatic ecological evaluation guidelines, regulations, and methods, some potential evaluation indicators are selected. Sensitive indicators specific to the Three Gorges Reservoir ecosystem are then identified by combining discriminant analysis and redundancy analysis. Finally, a multidimensional assessment system emphasizing aquatic habitat, aquatic organisms, and food web structure is constructed to evaluate the structural integrity of the aquatic ecosystem in the main stem of the Three Gorges Reservoir since the formation of the reservoir. The evaluation period is divided into three phases: the initial impoundment period (2003~2007), the 175 m pilot impoundment period (2008~2019), and the period of official impoundment operation (2020~2024). The evaluation results show that the overall structural integrity of the main stem of the Three Gorges Reservoir has gradually improved from poor to medium since the initial impoundment period up to now. The analysis results of three typical river sections, Zigui, Wanzhou and Fuling, showed that there was no significant difference in the aquatic ecosystem structural integrity among different river sections during the 175 m pilot impoundment period (2008~2019), all in a poor state. After the official impoundment operation (2020~2024), the evaluation grades generally improved by 1 to 2 levels, while the aquatic ecosystem structural integrity of Fuling and Wanzhou sections were better than that in Zigui section. The findings demonstrate that the implementation of comprehensive conservation initiatives, including the Yangtze River conservation, the ten-year fishing moratorium policy, and coordinated reservoir operation management, has yielded statistically significant ecological benefits and a measurable enhancement in the aquatic ecosystem structural integrity within the main channel of the Three Gorges Reservoir.