The Chishui River is a first-level tributary of the upper reaches of the Yangtze River with complex and diverse habitat types, playing an important role in the protection of rare and endemic fish resources in the upper reaches of the Yangtze River. In order to learn the health level of the Chishui River basin, a fish survey was conducted in 31 sites of the main stream and primary tributaries of the Chishui River in May and September 2020. A total of 6288 fish species belonging to 4 orders, 10 families, 26 genera and 90 species were collected. The value of Shannon-Wiener index ranged from 0.21 to 2.87 with an average value of 1.48±0.46, while those for Simpson and Pielou index were 0.08-0.91 with an average value of 0.69±0.14 and 0.13-0.97 with an average value of 0.76±0.13, showing a gradual increase trend from the upstream to downstream. The F-IBI index was adopted to assess the health status of Chishui River, the core index system of which containing 10 indicators, including total number of species, the number of cypriniformes %, the number of Gobioninae %, the number of Culterinae% etc. The F-IBI value of each sampling point was calculated by the ratio method, and five health grades were classified into ‘healthy（F-IBI≥5.99）’, ‘subhealthy（4.49≤F-IBI＜5.99）’, ‘average（2.99≤F-IBI＜4.49）’, ‘poor（1.50≤F-IBI＜2.99）’ and ‘extremely poor（F-IBI＜1.50）’ to evaluate the health status of each sampling point.In general, the surveyed sites showed healthy state, among which, 20 sites were in healthy status, 5 sites were in sub-healthy status, 3 sites were in general status, and 3 sites were in poor state. F-IBI values gradually increased from upstream to downstream, and presented significant differences in the upper, middle and downstream (P < 0.05). The main stream exhibited better health status than tributaries, the poor health sites are located in the Tongzi River and the Wuma River, while the general health sites are located in the Xishui River basin. Cypriniformes and Cyprinidae were found to be the dominant fish fauna composition in Chishui River, implying the F-IBI evaluation system was a good predictor of human disturbance and better reflected the health status of aquatic ecosystem in Chishui River, which can be used as a tool for aquatic ecosystem health evaluation and resource management for Chishui River.