Abstract: The rapid development of urbanization has led to changes in the morphology and physical habitats of urban rivers, the quality of water ecosystems has declined, and the water ecological safety of rivers has been seriously threatened, so monitoring and evaluating the water ecological safety of urban rivers plays an important role in guaranteeing the ecological protection of the watershed and the construction of urban ecological civilization. Taking the Qinhuai River basin as an example, we selected a typical urban river section as the research object, analyzed the community structure of aquatic organisms by using environmental DNA technology, and combined with the physical and chemical parameters of river water quality and changes in river physical habitats, we set up an ecological evaluation index system of the urban river to comprehensively assess the river's water ecosystem security status. The results show that this paper constructs a set of indicator system and assessment method for water ecological environment safety of urban rivers based on socio-economic impacts, water ecological health, ecological service function and regulatory management. Environmental DNA sequencing analysis showed that the phytoplankton, zooplankton and fish diversity indices in different areas of the Qinhuai River in Nanjing were high. However, the diversity index of benthos in the Qinhuai River was low, with a decrease in benthic species, miniaturization of individuals, and predominance of pollution-tolerant species. In addition, the economic impact index and pollution load index scores of the Qinhuai River Basin were lower in the comparative analysis. The grading of water ecological health index belongs to the status of “better”, the main influence factor is the poor water quality condition, and the comprehensive score of water quality is low. Comprehensive analysis of the Qinhuai River ecological risk is low, in a “safer” state. This paper builds a universal indicator library, and the method of assessment through indicator selection and weighting can be flexibly adjusted for rivers of different regions, grades, flows and ecological functions, and has wide applicability.