In the Wei River basin, the ecosystem is gradually deteriorating due to the rapid growth of the population and the development of economies. It is thus important to assess the ecosystem health and take measures to restore the damaged ecosystem. In this study, an index of biotic integrity (IBI) for fish was developed to aid the conservation of the ecosystem based on a calibration data set. An index of water and habitat quality (IWHQ) was calculated based on environmental variables and habitat quality (QHEI) to identify the environmental degradation in the studied sites. The least degraded sites (IWHQ ≤ 2; W1, W5, W10, W12, W13, W14, and W16) were chosen as the reference sites. Six metrics that are sensitive to environmental degradation were utilized to differentiate the reference and the impaired sites using statistical methods. These metrics included the number of species (P1), the total biomass (P2), the number of Cobitidae species (P8), the proportion of species in the middle and low tiers (P10), the proportion of individuals that were classified as sensitive species (P25), and number of individuals in the sample (P39). A continuous scoring method was used to score the six metrics, and four classes were defined to characterize the ecosystem health of the Wei River basin. The fact that the overall IBI scores were negatively correlated with the index of environmental quality (IWHQ) based on the validation data set indicated that the index should be useful for biomonitoring and the conservation of biodiversity. According to the results, more than half of the sites were classified as poor or very poor. The ecosystem health in the Wei River was better than that in the Jing River and the Beiluo River, and this study will be a great reference for water resources management and ecosystem restoration in the Wei River basin.