An accurate assessment of fish stocks is crucial for maintaining the health and stability of aquatic ecosystems. To understand the characteristics of fish diversity in the Qingshui River, this study utilized high-throughput sequencing data obtained through environmental DNA macrobarcoding technology (eDNA). The research explored differences in fish diversity and community characteristics in the Qingshui River by analyzing fish community structure, α diversity, β diversity, and the redundancy of environmental factors. This analysis aims to provide data support for water ecological monitoring and management. The results were as follows: (1) A total of 91 species of fishes from 18 families and seven orders were detected in the eDNA survey, and the dominant species was Cypriniformes, accounting for 72.5%. (2) There were significant differences in α diversity analysis in the Qingshui River (p < 0.05). (3) The fish community structure was similar in β diversity analysis. (4) In the redundancy analysis of environmental factors, pH, DO, TN, TP, NO2− and MnO4− have the greatest impact on the Qingshui River Basin. eDNA technology has the advantages of high efficiency and low harm and can be used for water ecological monitoring and management. It provides a scientific basis for fish protection and a valuable reference for fish monitoring in the future.