Abstract: The aims of this study are: (1) to detect fish diversity in Chongqing section of the national nature reserve of rare and endemic fishes in the upper Yangtze River by using environmental DNA metabarcoding (eDNA metabarcoding), (2) exploring new methods applicable to the monitoring and protection of fish diversity in the Yangtze River, (3) providing certain basic data for the evaluation of the effect of the “10-year ban on fishing in the Yangtze River” later. A total of 6 sampling points were set up in the Chongqing section of the reserve in March 2021. The fish diversity was detected by following procedures, water sample collection, eDNA capture and extraction, PCR amplification and sequencing, database comparison analysis and other environmental DNA metabarcoding standardized analysis. The results showed that 74 fish species were detected (excluding 3 genera that have not been identified at the species level), belonging to 6 orders, 16 families and 52 genera, including 2 national-level protected fish, 10 endemic fish in the upper reaches of the Yangtze River, 1 key protected fish in Chongqing, and 8 invasive species. The genus Cyprinus, Carassius, Ctenopharyngodon and Tachysurus were detected at each sampling site and became the dominant species in each site. The various indexes of Alpha and Beta diversity of fish at various points are relatively uniform, indicating that the ecological structure of fish in the reserve is relatively balanced and stable. In summary, this study showed that although environmental DNA metabarcoding cannot completely replace traditional fish resource monitoring methods, it is a good strategy to combine them to quickly investigate the diversity composition and distribution of fish species in the Yangtze River Basin.