Environmental DNA (eDNA) technology has emerged as a revolutionary tool for biodiversity monitoring in aquatic ecosystems, offering a non-invasive, cost-effective, and highly sensitive method for detecting species presence and abundance, particularly in complex and dynamic environments like the upper Yangtze River. In order to identify universal primers that are suitable for investigating fish diversity in the upper Yangtze River using eDNA technology, this study selected ten frequently used primers that amplify gene fragments from the 12S rRNA, 16S rRNA, Cytb, and COⅠ genes. The primers investigated were Mifish-U, AcMDB07, Teleo, 12SPv, Fish16S1, Ve16S1, PSI, G, VeCB1, and FishCB. DNA samples extracted from muscle tissues of 32 commonly encountered fish species in the upper Yangtze River and three additional aquatic species not from the upper Yangtze River were subjected to amplification using these primers. The results indicated that six primer pairs could amplify all 35 fish species, with Mifish-U showing the highest amplification efficiency. In addition, the eDNA samples from three sampling sites (Pingshan County, Fuling District, and Wushan County) in the upper Yangtze River were further subjected to high-throughput sequencing using the Mifish-U primer. A total of 80 fish species were detected, including the 32 fish species used in this study, with high discriminatory power. Moreover, through high-throughput sequencing, the Mifish-U primer was utilized to qualitatively and quantitatively analyze eDNA samples from indoor farms for three fish species. The results indicated a significant correlation (P<0.05) between the biomass of Pelteobagrus fulvidraco and Carassius auratus and the number of sequences, highlighting the great potential of the Mifish-U primer for eDNA quantitative analysis. In summary, our findings suggest that Mifish-U is more suitable as a universal primer for investigating fish diversity in the upper Yangtze River using eDNA technology. This study offers important insights into the selection of primers for monitoring fish diversity in the upper Yangtze River using eDNA technology.