Abstract: Urban development has altered the morphology and physical habitat of rivers, leading to a gradual deterioration of their water ecological environment. To systematically assess the security status of urban water ecological environment, this study focused on the characteristics of deteriorating water quality, sharp decline in aquatic organisms, and ecosystem degradation in urban rivers. Using the Qinhuai River in Nanjing City as a typical case, we investigated changes in the physiochemical parameters of river water quality and physical habitats. In conjunction with environmental DNA (eDNA) metabarcoding technology, we analyzed the community structure characteristics of aquatic organisms. An evaluation index system for the water ecological environment security of the Qinhuai River was constructed based on four major indicators: ecological environment pressure, water ecological health, ecological service function, and ecological risk, with a total of 19 sub-indicators. The results of eDNA sequencing showed that the dominant phytoplankton in the Qinhuai River were Cryptomonas, Ochrophyta, and Chlorophyta, accounting for 93.96%. The dominant zooplankton groups belonged to Ploima and Diplostraca, accounting for 63.37%. Among the benthic animals, Arthropoda and Cnidaria accounted for the highest proportion, reaching 98.8%. Among the fish, Cypriniformes were the dominant species, accounting for 69.99%. Based on the environmental DNA results, the diversity indices of phytoplankton, zooplankton, and fish in the aquatic ecosystem were relatively high, but the diversity index of benthic animals was low. In addition, the river's ecological health index was classified as "fair", primarily influenced by water quality and habitat complexity. The cumulative heavy metal pollution index for the river was 64.74, indicating a relatively high ecological risk for the Qinhuai River in Nanjing. The comprehensive analysis showed that the ecological security index of the Qinhuai River in Nanjing City was 59.39, indicating that the water ecological environment was in a "generally safe" state. This study provides a useful reference for the evaluation of urban river water ecological environment security by demonstrating a methodology for indicator selection and weight determination.