Extracellular vesicles (EVs) are nanoscale vesicles secreted by cells and play a pivotal role in intercellular communication. As crucial mediators in cell-to-cell signaling, EVs are instrumental in physiological and pathological processes. They serve not only as significant biomarkers in disease diagnosis but also hold promise as new drug and drug delivery system candidates due to their unique biological properties. The process begins with the cell membrane invagination to form a cup-like structure, selectively encapsulating surface proteins and soluble proteins to create early endosomes. Under the influence of the Endosomal Sorting Complex Required for Transport (ESCRT), Rab-GTPase, and tetraspanins, these early endosomes evolve into late sorting endosomes, which form multivesicular bodies. Upon fusion with the plasma membrane, these bodies release EVs into the extracellular space. EVs are internalized by target cells through ligand-receptor interactions, endocytosis, and membrane fusion, thereby executing biological functions. Endocytosis is a common uptake mechanism for EVs, with various pathways including clathrin-dependent pathways, caveolae-mediated uptake, macropinocytosis, phagocytosis, and lipid raft-mediated internalization. Once inside the recipient cell, EVs interact with the endosomal system, fuse, and release their contents into the cytoplasm. The absorption and distribution of EVs in the body are influenced by factors such as their origin, targeting, administration method, size, and surface characteristics. Through engineering, EVs can be loaded with specific proteins or RNA to achieve targeted drug delivery to specific organs or cells. In terms of disease diagnosis, the components of EVs can serve as biomarkers, offering new avenues for early detection, progression monitoring, and therapeutic efficacy assessment. They carry RNA and protein molecules that can reveal pathological changes in their originating cells. In terms of disease treatment, EVs have the potential for targeted delivery, serving as platforms for vaccine development and as drug delivery systems to transport drugs directly to specific cells or tissues. Moreover, EVs themselves can be used as therapeutic agents for autoimmune diseases and cancer. In the realm of EV separation and purification technology, common methods include ultracentrifugation, immunoaffinity chromatography, polymer co-precipitation, ultrafiltration, size exclusion chromatography, and microfluidics. However, due to the limitations of a single separation technique in meeting the demand for high-quality and high-purity EVs, multiple methods are often combined to separate and purify EVs effectively. This article concludes by summarizing the broad application prospects of EVs in the prevention and treatment of human diseases and highlights several key scientific questions that require further in-depth research. The potential of EVs in diagnostics and therapeutics, as well as the challenges in their isolation and characterization, underscores the need for continued exploration and innovation in this field.