It is crucial to gain valuable insights into the ecological health of rivers to inform management decisions and protect sustainable living conditions. Understanding biological information is vital to gaining insight into river ecosystem biodiversity, but reliable methods are challenging. Here, We investigate the potential impact of extracellular vesicles (EVs) in urban water systems on human and environmental health to promote urban sustainability. We used EXODUS to detect EVs and perform metaproteomic analysis on samples from an urban water system that contained human feces, wastewater, bacteria, plants, arthropods, and soil. We analyzed EVs collected from urban and green areas, observing taxonomic variations and discovering bacterial contributions to their protein content. According to our research, the abundance and expression levels of proteins in EVs can indicate how human activities affect microbial communities in rivers, potentially impacting public health. Our study offers a fresh perspective on the interconnectedness of urban sustainability, public health, and river ecosystem biodiversity.