Exchange of mobile functional genes within microbiota benefits the microbial community. However, the status of the mobile gene pool in environment is still largely unclear, impeding the understanding on the process of gene transfer in natural microbial communities. The release of extracellular vesicles (EVs) by diverse organisms has been proposed to be a vital way in the complex networks of interactions between microbes and their habitats. In this study, we hypothesized that microbial EVs encapsulating functional DNA are widely distributed in the environmental matrix. The prevalence, source and DNA cargoes of EVs in three types of typical microbial habitats were studied. High abundance of EVs comparable to the bacterial concentration was found in human faeces, wastewater and soil. Metagenomic analysis showed the diverse and differential taxonomy of EVs-associated DNA compared to source microbiome. An array of efficient EVs producing species was identified. A wide variety of mobile genes including glycoside hydrolase family 25 were enriched. Antibiotic resistance genes co-localizing with mobile genetic elements were abundant in the EVs. This study provides novel insights into the prevalent EVs as a reservoir for the mobile functional genes in the natural environment.