Poyang Lake is the largest freshwater lake in China, and its unique ecosystem plays an important role in maintaining biodiversity. However, the biodiversity of Poyang Lake is facing a serious threat as a result of human activities. Species investigation is the basis of biodiversity protection. In order to improve the water ecological monitoring system and achieve efficient and non-invasive species monitoring, environmental DNA metabarcoding was used in this study to assess biodiversity in different habitats in Poyang Lake. A total of 45 species including 31 fish species and six bivalve species were detected in water samples collected from 29 sampling sites in six habitats in Poyang Lake (Jiangxi section of the mainstream of the Yangtze River, channel connecting Poyang Lake and the Yangtze River, main lake area of Poyang Lake, Nanjishan Nature Reserve, Junshan Lake and Qinglan Lake). The species were detected through a standardized process involving water sample collection, filtration, environmental DNA extraction, genetic marker amplification, high-throughput sequencing and bioinformatics analysis. The 31 fish species, which included 11 alien species, were mainly cyprinids and lake dwellers. Alpha diversity analysis indicated a decline in biodiversity in Poyang Lake habitats and a serious need for water ecology conservation. Beta diversity analysis revealed significant differences in the biotic community structures of the six habitats in Poyang Lake. The results align with those obtained with more traditional methods, and hence, environmental DNA metabarcoding can be used as an alternative biodiversity monitoring tool for rapid detection of the diversity and spatial distribution of organisms (especially fish). This technique provides a new toolkit for biodiversity monitoring and aquatic ecological conservation in Poyang Lake area.