The article reviews the decade-long exploration of extracellular vesicle-associated DNA (EV-DNA) in human blood, highlighting its significance in intercellular communication and its potential as a biomarker for various diseases. Key advancements include the identification of EV-DNA in pancreatic cancer patients, the understanding of molecular mechanisms governing DNA emission, and the development of liquid biopsy strategies. The review covers the diverse mechanisms of EV-DNA emission, its functional roles, and the challenges in clinical application. It also discusses the potential of EV-DNA as a biomarker for cancer, tuberculosis, kidney injury, prenatal diagnosis, Parkinson’s disease, and inflammatory disorders. The article emphasizes the importance of standardized techniques for isolating and analyzing EV-DNA, and it outlines future directions for research, including the development of novel strategies for biomarker discovery and non-invasive disease detection.The article reviews the decade-long exploration of extracellular vesicle-associated DNA (EV-DNA) in human blood, highlighting its significance in intercellular communication and its potential as a biomarker for various diseases. Key advancements include the identification of EV-DNA in pancreatic cancer patients, the understanding of molecular mechanisms governing DNA emission, and the development of liquid biopsy strategies. The review covers the diverse mechanisms of EV-DNA emission, its functional roles, and the challenges in clinical application. It also discusses the potential of EV-DNA as a biomarker for cancer, tuberculosis, kidney injury, prenatal diagnosis, Parkinson’s disease, and inflammatory disorders. The article emphasizes the importance of standardized techniques for isolating and analyzing EV-DNA, and it outlines future directions for research, including the development of novel strategies for biomarker discovery and non-invasive disease detection.