Abstract: This study developed seasonal Zooplankton Index of Biotic Integrity (Z-IBI) frameworks to evaluate ecological health in the Chishui River Basin, based on zooplankton community data and water quality monitoring from 25 sampling sites across four seasons. Reference and impaired sites were identified using the Water Quality Index (WQI) and levels of anthropogenic disturbance. A total of 28 candidate biological metrics were selected based on zooplankton species number, abundance, biomass and community structure. These metrics underwent a stepwise screening process, including zero-value elimination, discriminatory power analysis based on the Indicator Quality (IQ) value, redundancy testing, and seasonal applicability evaluation. The results demonstrated that the Z-IBI effectively distinguished between reference and impaired sites (IQ≥2), while maintaining a consistent distribution of scores across the remaining sampling locations, indicating strong diagnostic performance for ecological health assessment. Overall, the Chishui River Basin showed generally good ecological conditions, although certain sites were identified as being at risk of degradation, with ecological health ratings classified as “poor” or “very poor.” Spatial analysis revealed no significant differences in Z-IBI scores between upstream and downstream regions (P>0.05). Compared with the WQI, the Z-IBI was more sensitive to ecological disturbances, capturing ecosystem changes that were not reflected by physicochemical indicators alone. The proposed Z-IBI framework provides a scientific basis for assessing the ecological health of small to medium-sized river basins and supports the identification of key areas requiring priority restoration and conservation.