Extracellular vesicles (EVs) have emerged as key players in intercellular communication, facilitating the transfer of crucial cargo between cells. Liquid biopsy, particularly through the isolation of EVs, has revealed a rich source of potential biomarkers for health and disease, including proteins and nucleic acids. A milestone in this exploration occurred a decade ago with the identification of extracellular vesicle-associated DNA (EV-DNA) in the bloodstream of a patient diagnosed with pancreatic cancer. Subsequent years have witnessed substantial advancements, deepening our insights into the molecular intricacies of EV-DNA emission, detection, and analysis. Understanding the complexities surrounding the release of EV-DNA and addressing the challenges inherent in EV-DNA research are pivotal steps toward enhancing liquid biopsy-based strategies. These strategies, crucial for the detection and monitoring of various pathological conditions, particularly cancer, rely on a comprehensive understanding of why and how EV-DNA is released. In our review, we aim to provide a thorough summary of a decade's worth of research on EV-DNA. We will delve into diverse mechanisms of EV-DNA emission, its potential as a biomarker, its functional capabilities, discordant findings in the field, and the hurdles hindering its clinical application. Looking ahead to the next decade, we envision that advancements in EV isolation and detection techniques, coupled with improved standardization and data sharing, will catalyze the development of novel strategies exploiting EV-DNA as both a source of biomarkers and therapeutic targets. EVs and NVEPs encapsulate an array of bioactive molecules crucial for cell-cell communication in both physiological and pathological contexts. Recent comprehensive reviews and studies have highlighted distinctive variations in proteomic, nucleic acids, and lipid content between NVEPs and various EV subpopulations. While considerable attention has been directed towards certain bioactive molecules, such as proteins and RNA, the exploration of EV-DNA, including mitochondrial DNA (mtDNA), has remained relatively under-explored despite its discovery in human blood more than a decade ago. Although research in this domain has been limited, there is an increasing recognition of the biological significance of EV-DNA. Emerging evidence underscores its potential as a source of biomarkers and diagnostic candidates in diverse pathological conditions, including cancer, tuberculosis, kidney injury, prenatal diagnosis, Parkinson's disease, and inflammatory disorders. In this review, we highlight the putative molecular mechanisms of EV-DNA emission and summarize the current knowledge on EV-DNA function in human health and disease, including intercellular communication, cancer progression, and immune regulation. Collating a decade's worth of research, we delve into the potential clinical and diagnostic applications of EV-DNA, emphasizing its role in biomarker discovery and non-invasive disease detection and monitoring. Furthermore, we summarize the most common techniques used to characterize the genomic and epigenetic content of EV-DNA. Lastly, we assess the potential functional roles of EV-DNA and its impact on human health and disease.Extracellular vesicles (EVs) have emerged as key players in intercellular communication, facilitating the transfer of crucial cargo between cells. Liquid biopsy, particularly through the isolation of EVs, has revealed a rich source of potential biomarkers for health and disease, including proteins and nucleic acids. A milestone in this exploration occurred a decade ago with the identification of extracellular vesicle-associated DNA (EV-DNA) in the bloodstream of a patient diagnosed with pancreatic cancer. Subsequent years have witnessed substantial advancements, deepening our insights into the molecular intricacies of EV-DNA emission, detection, and analysis. Understanding the complexities surrounding the release of EV-DNA and addressing the challenges inherent in EV-DNA research are pivotal steps toward enhancing liquid biopsy-based strategies. These strategies, crucial for the detection and monitoring of various pathological conditions, particularly cancer, rely on a comprehensive understanding of why and how EV-DNA is released. In our review, we aim to provide a thorough summary of a decade's worth of research on EV-DNA. We will delve into diverse mechanisms of EV-DNA emission, its potential as a biomarker, its functional capabilities, discordant findings in the field, and the hurdles hindering its clinical application. Looking ahead to the next decade, we envision that advancements in EV isolation and detection techniques, coupled with improved standardization and data sharing, will catalyze the development of novel strategies exploiting EV-DNA as both a source of biomarkers and therapeutic targets. EVs and NVEPs encapsulate an array of bioactive molecules crucial for cell-cell communication in both physiological and pathological contexts. Recent comprehensive reviews and studies have highlighted distinctive variations in proteomic, nucleic acids, and lipid content between NVEPs and various EV subpopulations. While considerable attention has been directed towards certain bioactive molecules, such as proteins and RNA, the exploration of EV-DNA, including mitochondrial DNA (mtDNA), has remained relatively under-explored despite its discovery in human blood more than a decade ago. Although research in this domain has been limited, there is an increasing recognition of the biological significance of EV-DNA. Emerging evidence underscores its potential as a source of biomarkers and diagnostic candidates in diverse pathological conditions, including cancer, tuberculosis, kidney injury, prenatal diagnosis, Parkinson's disease, and inflammatory disorders. In this review, we highlight the putative molecular mechanisms of EV-DNA emission and summarize the current knowledge on EV-DNA function in human health and disease, including intercellular communication, cancer progression, and immune regulation. Collating a decade's worth of research, we delve into the potential clinical and diagnostic applications of EV-DNA, emphasizing its role in biomarker discovery and non-invasive disease detection and monitoring. Furthermore, we summarize the most common techniques used to characterize the genomic and epigenetic content of EV-DNA. Lastly, we assess the potential functional roles of EV-DNA and its impact on human health and disease.