Extracellular vesicles (EVs) are nanoscale membrane-bound vesicles released by cells into bio-fluids. EVs carry diverse molecular cargoes which enable them to mediate intercellular communication. These molecular cargoes comprise inherited constituents such as nucleic acids and proteins, as well as surface modifications and membrane-associated molecules, which can serve as biomarkers for minimally-invasive disease detection. In this chapter, we first describe the biogenesis of EVs, through which molecular information can be passed onto EVs, thereby enabling EVs as disease biomarkers, as well as elaborate on the potential use of EV biomarkers for disease diagnosis. Finally, to overcome the technical challenges of conventional analytical platforms in detecting EVs, we present several new technologies optimised for the profiling of nanoscale EV features from bio-fluids for diagnostic applications.