To explore the barrier effects of cascade dams on different fish species in the main tributaries of the upper reaches of the Yangtze River, the present study has analyzed fish species composition and diversity across 9 sections of the Qijiang River using environmental DNA (eDNA) technology in May 2021. These findings were compared with the results using traditional fisheries investigation methods, and the potential application of eDNA technology in Yangtze River system fish monitoring was explored. The results showed that a total of 59 fish species were detected by eDNA technology, which were categorized into 7 orders, 16 families, and 49 genera. Among these fish species, 41 species (accounting for 69.49%) belonged to the Cypriniformes, and 12 species (accounting for 20.34%) were the rare-endemic ones in the upper reaches of the Yangtze River. A total of 92 fish species were captured by traditional fisheries investigation methods, which belonged to 7 orders, 17 families, and 64 genera. The comprehensive analysis results of the two methods suggested that 96 fish species inhabited the Qijiang River. Among them, 55 species (accounting for 57.29%) were obtained by both methods. Moreover, from downstream to upstream of the Qijiang River, the 59 fish species detected by eDNA technology showed the following trends: (1) the diversity of fish species in different sections exhibited a step-down decreasing trend; (2) the similarity of fish species between each sampling site and the entire basin exhibited a stepwise decreasing trend; (3) The ratio of species quantity between the fish preferring flowing water and those preferring calm and slow flowing water decreased gradually, additionally, both the number and proportion of migratory fish species gradually decreased; (4) as the proportion of bottom fish species gradually decreased, the proportion of pelagic and lower-middle fish species gradually increased. Hence, these findings demonstrated that the prolonged barrier created by dams had a substantial impact on the composition and diversity of fish species in the Qijiang River, with cascade dams exerting more pronounced effects compared to single dams. The cascade dams had obvious barrier effects on the flowing, migratory, and bottom fish species (including the rare and endemic ones in the upper reaches of Yangtze River). Finally, under the existing research basis and background conditions, we believe it is applicable for eDNA technology to be used in fish investigation of the Yangtze River system if the local database for species annotation is adequately improved. Otherwise, this technology is suggested to be properly combined with traditional fisheries investigation methods to improve the comprehensiveness and credibility of the final investigation results. The results of present study will be helpful for understanding the status of fishery resources and the barrier effects of cascade dams on different fish species in the Qijiang River. Meanwhile, the application prospects of eDNA technology will provide scientific and technical supports for protection of fishes in the Yangtze River.