In 2020, Environmental DNA Metabarcoding was used to study the fish diversity in the Huangjinxia section of the upper Hanjiang River, and the historical survey data were compared to construct a fish list in the Huangjinxia section of the upper Hanjiang River. A total of 20 fish species were detected from 9 sampling sites, accounting for 45.45% of the list; Cyprinus_carpio, Carassius_auratus, Distoechodon_tumirostris and Squalidus_argentatus are the dominant species. The Shannon index of fish in the upper and lower reaches of the Huangjinxia was significantly different (P < 0.01, n=9), and the Shannon index of fish in the upper reaches was significantly larger than that in the lower reaches. The main reason for the spatial differences in fish diversity is the Huangjinxia hydraulic hub. The fish composition detected by environmental DNA was similar to that obtained by traditional methods. As an emerging biomonitoring tool, environmental DNA macrobarcoding technology can complement traditional fish monitoring methods to rapidly detect fish diversity and its spatial distribution in the upper reaches of the Hanjiang River.