Extracellular vesicles (EVs) are diverse, nanoscale membrane vesicles actively released by cells. Initially considered cellular debris, EVs are now recognized as important vehicles for intercellular communication and circulating biomarkers in disease diagnosis and prognosis. However, the lack of sensitive preparatory and analytical technologies poses a barrier to their clinical translation. This article reviews recent advances in EV research, focusing on new analytical platforms and their clinical applications. EVs are classified into exosomes, microvesicles, and apoptotic bodies based on their biogenesis. Exosomes are produced through endosomal membrane invagination, while microvesicles are derived from outward blebbing of the plasma membrane. EVs contain a variety of cellular cargos, including proteins, RNAs, and lipids, which can be used as biomarkers for disease detection and monitoring. Physical characterization methods such as microscopy, dynamic light scattering, and nanoparticle tracking analysis are used to measure EV size, distribution, and concentration. New enrichment methods, including microfluidic filtering, contact-free sorting, and immunoaffinity capture, improve isolation efficiency and specificity. Protein analysis techniques, such as western blotting, ELISA, and mass spectrometry, are crucial for identifying physiological and pathological markers. New protein analysis methods, including small particle flow cytometry, micro-nuclear magnetic resonance, nano-plasmonic exosome sensors, and integrated magnetic-electrochemical exosome sensors, offer improved sensitivity and miniaturization for point-of-care applications. These advancements are expected to have a significant impact on both basic and translational studies of EVs.Extracellular vesicles (EVs) are diverse, nanoscale membrane vesicles actively released by cells. Initially considered cellular debris, EVs are now recognized as important vehicles for intercellular communication and circulating biomarkers in disease diagnosis and prognosis. However, the lack of sensitive preparatory and analytical technologies poses a barrier to their clinical translation. This article reviews recent advances in EV research, focusing on new analytical platforms and their clinical applications. EVs are classified into exosomes, microvesicles, and apoptotic bodies based on their biogenesis. Exosomes are produced through endosomal membrane invagination, while microvesicles are derived from outward blebbing of the plasma membrane. EVs contain a variety of cellular cargos, including proteins, RNAs, and lipids, which can be used as biomarkers for disease detection and monitoring. Physical characterization methods such as microscopy, dynamic light scattering, and nanoparticle tracking analysis are used to measure EV size, distribution, and concentration. New enrichment methods, including microfluidic filtering, contact-free sorting, and immunoaffinity capture, improve isolation efficiency and specificity. Protein analysis techniques, such as western blotting, ELISA, and mass spectrometry, are crucial for identifying physiological and pathological markers. New protein analysis methods, including small particle flow cytometry, micro-nuclear magnetic resonance, nano-plasmonic exosome sensors, and integrated magnetic-electrochemical exosome sensors, offer improved sensitivity and miniaturization for point-of-care applications. These advancements are expected to have a significant impact on both basic and translational studies of EVs.