This study aims to advance Korea’s aquatic ecosystem assessment framework by developing and validating an Integrated Assessment Index (IAI) that synthesizes three biological indicators: the Diatom Assessment Index (DAI), the Benthic Macroinvertebrate Assessment Index (BAI), and the Fish Assessment Index (FAI). Using biomonitoring data collected from 2019 to 2021, three integration methods: the minimum grade method, most frequent grade method, and arithmetic mean method, were compared. The arithmetic mean method demonstrated the highest suitability and was adopted as the final integration approach. The resulting IAI showed stronger correlations with major water quality factors (BOD, TN, TP) than individual biological indices, indicating its enhanced capacity to capture both water quality gradients and ecological response patterns across biological assemblages. Application of the IAI to aquatic ecosystem assessment data from 2016 to 2023 revealed that the overall ecological condition of Korean rivers remained at a “fair (C)” level. Approximately half of the sites were classified as good to very good (A~B), while around 20% were rated as poor to very poor (D~E). Annual cycle analysis further indicated that first-year surveys within each monitoring phase exhibited higher proportions of good conditions, whereas second- and third-year surveys showed increasing frequencies of fair conditions, suggesting cumulative environmental stressors or progressive habitat alteration. Mid-sized basin target standards achievement analysis showed a clear discrepancy between waterquality and biological outcomes. While BOD and TP targets were met at relatively high rates, IAI achievement rates were the lowest across all major river basins (18~33%). This indicates that current water quality centered management goals insufficiently reflect actual ecological conditions. The results highlight that improvements in physico-chemical factors alone are insufficient for biological recovery and that habitat structure, flow regime, and substrate conditions are critical drivers of ecological integrity. Overall, the IAI effectively integrates biological and physico-chemical information, offering a more comprehensive quantification of river aquatic ecosystem health than single metric approaches. The index demonstrates strong potential as a practical tool for future policy applications, including mid-sized basin target management, ecological restoration prioritization, and integrated water resource planning.