Extracellular vesicles (EV) are highly heterogeneous nanoscale vesicles secreted by cells. They carry various bioactive molecules derived from the parent cells. EV are widely distributed in various body fluids, showing enormous potential in liquid biopsy and disease treatment. However, conventional flow cytometers face challenges in detecting single EV with a diameter smaller than 300 nm. The nano-flow cytometry (nFCM) developed based on Raleigh scattering and sheath-flow single-molecule fluorescence detection has successfully pushed the detection limit of EV to 40 nm. Through multi-parameter detection at the single-particle level, nFCM enables simultaneous analysis of particle size, particle concentration, and multiple biochemical properties of individual EV. nFCM can be applied to clinical diagnosis and therapeutics based on EV.