Phytoplankton diversity is closely related to environmental variables and has been widely used in ecological health assessment of rivers and lakes. Combining the advantages of DNA-based identification and high-throughput sequencing technology, environmental DNA (eDNA) metabarcoding permits a new measurement for biodiversity monitoring in aquatic ecosystems. However, it had rarely been used to explore the variability and similarity of phytoplankton diversity between a lake and its inflow rivers and the effects of environmental variables on phytoplankton. This study utilized eDNA metabarcoding to investigate the spatial distribution of phytoplankton and the impacts of environmental variables on the phytoplankton diversity in Dianchi Lake (one of the most polluted urban lakes in China) and its main inflow rivers (Panlong River, Baoxiang River and Chai River).  The OTU table is  the original table about phytoplankton taxa. A total of 243 distinct phytoplankton taxa were detected, covering 9 phyla, 30 classes, 84 orders and 132 families, and the taxonomic richness of rivers was higher than that of Dianchi Lake.