Extracellular vesicles (EVs) are nanoscale lipid membrane–bound vesicles that are secreted by cells of both prokaryotes and eukaryotes and carry bioactive cargos including proteins, nucleic acid and lipids from source cells. Given their prominent ability in transporting bioactive components, EVs are regarded as promising biomarkers for disease diagnosis and emerging therapeutic nanoparticles. However, to exert their effect in clinical applications, effective isolation and sensitive analysis of EVs from complex biofluids is required. Recent advances in EV-related research has provided feasible approaches for developing emerging therapeutic nanoplatforms using EVs. With this review, we aim to provide a comprehensive and in-depth summary of recent advances in diverse assay methods for EVs including fluorescence, Raman/Surface-enhanced Raman Spectroscopy (SERS) analysis and other methods, as well as their clinical potential in constructing EV-based theranostic nanoplatforms towards various diseases. In particular, microfluidic-assisted analysis sytems, single EV detection and the main approaches of utilizing EVs for therapeutic purposes are highlighted. We anticipate this review will be inspirational for researchers in related fields and will provide a general introduction to scientists with various research backgrounds.