River ecosystem health assessments are a hot topic in global water resources management. The Huai River Basin is a representative basin faced with serious environmental problems due to high water pollution and numerous water projects. Comprehensive investigation of the factors affecting river ecosystem health is an urgent precursor to the successful management of the Huai River Basin. Based on a series of aquatic ecosystem indicators, monitoring and standards, such as phytoplankton, zooplankton and benthic macroinvertebrate data, an assessment framework of river ecosystem health is proposed using a multi-step gray correlation evaluation method. Environmental impact factors including climate, land cover, edaphic factors, hydrology, water projects and water quality were identified using the Kruskal-Wallis test and distance-based redundancy analysis method. The ecosystem was found to be sub-healthy with 44% (31/71) of sites in a healthy condition, 51% (36/71) sub-healthy and 5% (4/71) unhealthy. Climate and hydrology were the dominant factors that together contributed 37.5%, 24.1% and 6.9% of the variation in phytoplankton, zooplankton and benthic macroinvertebrates, respectively. The main variables affecting variation were minimum temperature, relative humidity, solar radiation, and the magnitude, frequency, duration and timing of monthly runoff. Together, these variables