Abstract: The Bao’an Lake is a representative medium-sized lake among the Jianghan Lake Group. This lake, disconnecting to the river decades ago, have rich biological bait resources and good ecological environment. As important baits of fish, zooplankton is the key component of material and energy circulation in lake ecosystem, and good indicator of ecological health condition. The zooplankton diversity in Bao’an Lake was studied in the 1990s, however, the current status is unclear. In recent years, the rapid development of environmental DNA and metagenomic techniques provided a new approach to study the zooplankton diversity. In this study, water samples were collected from Bao’an Lake in the four seasons in 2019. Based on metagenomic sequencing, we optimized species identification and abundance estimation based on the metagenomic data, and subsequently analyzed the diversity of zooplankton in Bao’an Lake. The results showed that: (1) 374 OTUs of zooplankton were identified, including 282 protozoa, 45 cladocera, 26 copepoda and 21 rotifera, respectively; (2) For different seasons, high zooplankton diversity was found in summer and autumn; for different lake areas, high zooplankton diversity have been found in the Main-Lake-area and Xiao-Sihai-area; (3) Seasonal changes had greater effect on zooplankton community structure than different lake areas; (4) Temperature and chlorophyll-A were the main environmental factors affecting zooplankton community structure in Bao’an Lake. Five patterns have been found in correlations between the abundance of OTUs and environmental factors. Among them, the dominant groups of protozoa are Ciliophora and Cercozoa, their abundance correlate with the levels of nitrate nitrogen, chemical oxygen demand, and temperature, while Cladocera and Copepoda have similar relationship with environmental factors, and show significant correlations between chlorophyll-A and orthophosphate. In summary, we investigated the zooplankton diversity in Bao’an Lake using metagenomic approach, and provided the basic data for understanding the biodiversity changes in Bao’an Lake.