Abstract: To assess the ecological health status of rivers in the Dongliangzhu River Basin of Songhuajiang River, 20 sampling sites were deployed across the basin. Macrobenthos community surveys were conducted in July (summer) 2023, October (autumn) 2023, and April (spring) 2024. Through analyses of the distribution range, discriminant ability, and correlation of 27 candidate indicators, three core indicators were finally selected to construct the evaluation system for the benthic-index of biotic integrity (B-IBI), namely the total number of taxa, the number of Plecoptera taxa, and the Biotic Index (BI). The ratio method was used to unify the dimension of each biological index, and the B-IBI value was obtained by summing the scores of individual indices. Based on the 25th percentile of B-IBI values at reference sites, the health assessment criteria for the Dongliangzhu River were ultimately established. Among the 20 sampling sites, 4 were classified as "healthy", 4 as "sub-healthy", 4 as "average", 5 as "poor", and 3 as "very poor". The average B-IBI score was 1.39, which fell into the "average" category according to the established assessment criteria. Water physicochemical indicators were identified as key environmental factors affecting the B-IBI assessment results of the Dongliangzhu River, with total phosphorus (TP), total nitrogen (TN), ammonia nitrogen (NH₃-N), nitrate nitrogen (NO₃⁻-N), nitrite nitrogen (NO₂⁻-N), permanganate index (COD_Mn), dissolved oxygen (DO), and electrical conductivity (EC) showing significant correlations with B-IBI values. Additionally, land use types had significant impacts on both the B-IBI assessment results and water physicochemical indicators of the basin, among which the area proportions of forestland and cropland were the critical land use type indices. Furthermore, human activity disturbances, as important drivers of land use changes, were also the main environmental factors leading to variations in the B-IBI assessment results of the Dongliangzhu River.