Poyang Lake is the largest freshwater lake in China, and its unique ecosystem is an important biodiversity reservoir. Fish play an important role in the aquatic ecosystem of Poyang Lake. Accurate assessment of fish species diversity and community composition is essential for studies of fish ecology and conservation management. In this study, environmental DNA metabarcoding was used to analyze fish diversity and community structure in different habitats and seasons in the Poyang Lake Basin. A total of 52 fish species were identified, and limnicolous fish were the most abundant; most fish were omnivorous and small. The fish diversity index was highest in the spring and in the main lake area of Poyang Lake. Cluster analysis and non-metric multidimensional scaling analysis revealed significant differences in fish community structure among habitats. Water temperature and pH were the main environmental factors affecting fish community structure in the Poyang Lake Basin. Non-invasive environmental DNA monitoring methods could provide an important auxiliary method for monitoring fishery resources during the 10-year fishing ban period. This study provides basic information on the fish resources in the Poyang Lake Basin and has implications for fishery management and the development of ecological protection policies.