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In this study, we evaluated the aquatic ecosystem health (AEH) with five grades (A; very good to E; very poor) of FAI (Fish Assessment Index), TDI (Trophic Diatom Index), and BMI (Benthic Macroinvertebrate Index) using the results of SWAT (Soil and Water Assessment Tool) stream water temperature (WT) and quality (T-N, T-P, NH4, NO3, and PO4). By applying Random Forest, one of the machine learning algorithms for classification analysis, each AEH index was trained and graded from the SWAT results. For Han river watershed (34,418 km(2)) in South Korea, the 8 years (2008 similar to 2015) observed AEH data of Spring and Fall periods at 86 locations from NAEMP (National Aquatic Ecological Monitoring Program) were used. The AEH was separately trained for Spring (FAI(s), TDIs, and BMIs) and Fall (FAI(a), TDIa, and BMIa), and the AEH results of Random Forest with SWAT (WT, T-N, T-P, NH4, NO3, and PO4) as input variables showed the accuracy of 0.42, 0.48, 0.62, 0.45, 0.4, and 0.58, respectively. The reason for low accuracy was from the weak strength of the individual trees and high correlation between the trees composing the Random Forest due to the data imbalance. The AEH distribution results showed that the number of Grade A of total FAI, TDI, and BMI were 84, 0, and 158 respectively and they were mostly located at the upstream watersheds. The number of Grade E of total FAI, TDI, and BMI were 4, 50, and 13 and they were shown at downstream watersheds.