Extracellular vesicles (EVs) were isolated from the culture supernatant oflung adenocarcinoma A549 cells. The size ofEVs was characterized as approximately 100 nm via DLS and TEM analysis, with the EV-specific marker TSG101 detected in the samples. The extracted EV DNA (evDNA) showed a predominantly～ 12 kb size distribution as assessed by agarose gel electro- phoresis. Based on sequencing data, specific evDNA was selected for PCR detection. DNase I or PS nuclease treatment ofthe EVs confirmed that the evDNA existed on the outside ofthe vesicle in a linear manner, and poly-dA tails were added to evDNA by TdT enzyme treatment, allowing for their capture by oligo-dT beads and presenting further evidence oftheir linear surface distribution. Additionally, a TetO-DNA/TetR-GFP visualization system was established to confirm the presence ofTetO-DNA in the EVs, which could be visualized byTetR-GFP proteins under fluorescent microscopy. Enrichment ofEVs carrying the TetO-DNA was confirmed via flow cytometry and PCR experiments using TetR-GFP protein. These findings collectively suggest that evDNA is distributed linearly on the surface ofEVs, providing a basis for further research into their biological functions.