The lack of freshwater ecosystem damage assessment system seriously hinders the implementation of environmental justice and ecological compensation in China. To address this gap, we developed a comprehensive freshwater ecosystem damage assessment system, guided by national standards for ecological environment damage assessment and the practical needs of environmental protection public interest litigation. The system encompasses three dimensions, including environmental quality, biological integrity and ecological function, and is structured into 1 target, 2 sub-targets, 8 criteria, and 27 indicators. In this system, the ecological baseline of the indicators is calculated by the trisection method and the quartile method, and hierarchical calculation formulas from the indicator to the target were provided. The degree of ecosystem damage is measured by the ecological damage index (EDI), with threshold values of 1.2, 1.5, and 2.0 representing mild, moderate, and severe damage, respectively. To validate the system’s applicability, case studies were conducted on Honghu Lake and a river in Xianning. To further improve the effectiveness of the system, the sensitivity and evaluation accuracy of the criterion factors was analyzed. Ensuring evaluation accuracy, we developed a prioritized combination list of assessment criteria (R2 > 0.9, RMSE < 0.1). Subsequently, non-core indicators in the same criterions were removed through correlation analysis. Finally, a prioritized combination list of 1 to 4 levels was provided, including 8, 7, 6, and 5 criteria, respectively, covering 14 to 21 indicators. The construction and application of this freshwater ecosystem damage assessment system not only advance the development of water ecological damage assessment theory in China, but also provide robust theoretical and methodological support for ecological environment protection law enforcement and ecological compensation practices.