Qinhuai River is the mother river of Nanjing, and its biodiversity is seriously threatened by the process of urbanization. The survey of species resources is the basis of biodiversity protection. Compared with morphological monitoring, the environmental DNA metabacroding is a simple, efficient and sensitive new monitoring technology. In order to explore the biodiversity of plankton, zoobenthos and fish in Qinhuai River, the environmental DNA metabacroding was used to explore it in July 2019, the differences between upstream and downstream of Qinhuai River were analyzed, and the relationship between the community composition of Qinhuai River and environmental variables was evaluated. The results showed that a total of 13 genus, 22 species and 407 operational taxonomic units (OTUs) of zooplankton, 85 gennus, 60 species and 4445 OTUs of phytoplankton, 16 genus, 17 species and 212 OTUs of zoobenthos, 53 genus, 44 species and 1663 OTUs were detected through the environmental DNA metabacroding. Among them, Ploima and Diplostraca, accounting for 63.37%, were dominant species in zooplankton community at the order level. The most abundant species in phytoplankton community at the phylum level were Cryptomonas and Ochrophyta, accounting for 88.11%. The zoobenthos were dominated by Arthropoda at the phylum level, which accounted for 91.67%. The Cypriniformes accounting for 69.99%, were dominants in fish of different samples at the order level. Compared with the historically morphological monitoring data of Qinhuai River, the environmental DNA metabarcoding technique was more powerful than the morphological method in assessment of species richness. Through the principal coordinate analysis (PCoA) and PERMANOVA test, a significant difference (P < 0.001) was found among downstream, the south branch of upstream and the north branch of upstream of Qinhuai River. Zooplankton, phytoplankton and zoobenthos were more affected by the group of sites than fish. Alpha(α) diversity of downstream in Qinhuai River was poorer than that of upstream, and Alpha(α) diversity of the south branch of upstream (Nanjing) was richer than that of the north branch (Jurong). All of the taxa of Qinhuai River except fish showed strong relationship between geographical distance (km) and biotic community similarity based on Bray-Curtis distance. Redundancy analysis (RDA) showed that organisms with lower trophic levels were more sensitive to changes in the environmental factors. The main influencing factors in plankton and zoobenthos community distribution were total nitrogen (TN), total organic carbon (TOC), total phosphorus (TP), ammonia nitrogen (NH₃-N), chemical oxygen demand (COD) and dissolved oxygen (DO). While the key factors in fish community distribution were DO, TOC, TN, TP and COD. Our results can provide a theoretical reference for the protection of the biodiversity of Qinhuai River.