Due to the interference of strong human activities, the ecological problems of urban lakes have become increasingly prominent. As a high-level consumer of the aquatic food chain, fish is an important part of the aquatic ecosystem and an important indicator of the health of aquatic ecosystems. In this study, ten urban lakes in Wuhan and its surrounding cities were selected as study areas. Fish data monitored by environmental DNA (eDNA) were used to analyze the characteristics of fish communities and establish a fish-based index of biotic integrity (F-IBI). The health status of lakes were evaluated and studied through F-IBI evaluation system targeting the ecological health of urban lake water bodies. The research revealed that during the survey periods, there were 50 fish species belonging to 39 genera, 10 families, and 7 orders. Among them, 7 species, including Hemiculter bleekeri, Hypophthalmichthys nobilis, Rhinogobius cliffordpopei, Rhinogobius giurinus, Cyprinus carpio, Megalobrama amblycephalaandCarassius auratus were identified as dominant species. At the order and family levels, Cypriniformes and Cyprinidae fish were the dominant groups. The F-IBI evaluation result showed that among all the 27 sampling sites in ten lakes, 11 sites were evaluated being health, accounting for 40.74%; 4 sites being sub-health, accounting for 14.81%; 10 sites being general, accounting for 37.04%; 2 sites being poor, accounting for 7.41%. Analysis based on the lake water quality data showed that the evaluation results reasonably reflected the health of the different urban lake ecosystem. The study indicated that the eDNA-based F-IBI method performs well in ecosystem health assessment of urban lakes. The research results provided strong basic supports for guiding the precise ecological restoration and ecosystem management.