Abstract: To assess the health status of aqua-system of Cao′e River, fish resources in nine sampling points in the main stream and tributaries of Cao′e River were investigated from 2016 to 2018, and the fish-based index of biotic integrity (F-IBI) was established. A total number of 17 116 individuals of fishes were collected, which belonged to 12 orders, 25 families, 69 genera and 107 species. Twenty-three candidate indices in five properties, including species composition and richness, nutritional structure, environmental tolerance, reproductive colocation group, fish quantity and health status, were proposed to develop the F-IBI. The results of data analysis show that four indices, i.e. species number of Cypriniformes (M3), ratio of species number in middle and lower layer (M9), ratio of sensitive fish individuals (M15) and ratio of species number spawned eggs in Mollusca (M19), were screened out as the core indices. Meanwhile, the scoring standard with diagnostic significance for fish biotic integrity was established and successfully used to evaluate the health statuses of nine sampling points in Cao′e River basin. The results show that the health statuses of the nine sampling points along the main stream were in "very poor-poor" to "normal" levels, while those of the tributaries were in "normal" to "good" levels, indicating that the health statuses of the tributaries were better that those of the main stream. The results suggest that, water pollution, obstruction of water conservancy projects and overfishing might be the important factors causing the lower health statuses of the main stream of Cao′e River, and water conservancy projects might be the important factor causing the lower health status of the main tributary Changzhao.