The absence of a standardized damage assessment system for freshwater ecosystems significantly impedes the implementation of environmental justice and ecological compensation mechanisms in China. To address this gap, we developed a comprehensive assessment framework based on national technical guidelines for ecological and environmental damage assessment and the practical requirements of public interest litigation for environmental protection. This system integrates three dimensions—environmental quality, biological integrity, and ecological function—and is structured hierarchically into 1 overall objective, 2 sub-objectives, 8 criteria, and 27 indicators. Ecological baselines for each indicator were established using the trisection and quartile methods, with hierarchical formulas enabling stepwise aggregation from indicators to the overall objective. Ecosystem damage severity is quantified using an ecological damage index (EDI), with thresholds of 1.2, 1.5, and 2.0 corresponding to mild, moderate, and severe damage, respectively. The system was validated through case studies in Lake Honghu and a river in Xianning, Hubei Province. To enhance evaluation efficiency, sensitivity and accuracy analyses of the criteria were conducted, leading to the development of an optimized set of assessment criteria combinations with high accuracy (R²＞0.9, RMSE＜0.1). Non-core indicators within each criterion were subsequently eliminated through correlation analysis, resulting in a prioritized four-level combination scheme comprising 8, 7, 6, and 5 criteria, encompassing 13 to 21 indicators in total. The establishment and application of this assessment system not only advance the theoretical foundation for aquatic ecological damage assessment in China but also provide robust methodological support for ecological and environmental law enforcement and compensation practices.