Abstract: Biological integrity has been widely used recently in river health assessment. The selection of the reference condition and the impacted condition are the most critical components in the process of developing an index of biological integrity. Meanwhile, the index of biological integrity is highly correlated with river type or eco-region in the watershed. For the purpose of developing a fish index of biological integrity (F-IBI), 32 sampling sites in the Hun-Tai River were sampled during the spring-summer period in 2014. Considering the natural variation of fish communities from upstream to downstream, 32 sample sites were classified into two types:the upper-middle streams and rivers according to species composition of fish assemblages and local fresh water eco-region delineation studies. All the reference sites and impaired sites were carefully selected based on standardized criteria, considering water quality and habitat quality. The results showed that the core indicators of F-IBI varied between upper-middle streams and rivers. Species richness, Shannon-Wiener's diversity index, species percentile of Cypriniforms, Leuciscinae, Cobitidae, Perciformes and omnivores, individuals percentile of Leuciscinae and intolerant species were selected as the core indicators in the upper-middle streams. Species richness, Shannon-Wiener's diversity index, species percentile of Gobioninae, Perciformes, Gobiidae, species inhabiting the surface layer, northeast China endemic fish, and invertivores, individuals percentile of tolerant species were selected as the core indicators in the rivers. Five categories of river health condition including excellent, good, fair, poor and very poor were used in the present study. The biological integrity results showed that the Hun-Tai river was generally degraded. 37.5% of sampling sites were under poor or very poor conditions, 21.88% of sampling sites were under fair conditions, and only 6.25% of sampling sites were under “excellent” conditions. Pearson's correlation analysis showed that our F-IBI is a useful and reliable index in the Hun-Tai River. F-IBI was significantly negative correlated with ρ(BOD₅), ρ(COD_Cr), ρ(NH₃-N), ρ(TN), and significantly positively correlated with the integrated habitat assessment index (IHAI).