Extracellular vesicles (EVs), one of the basic ways of intercellular interactions, carry a large number of biological substances and show great potential in disease diagnosis and treatment. Compared with tissue biopsy, its detection has the advantages of non-invasive, convenient sampling and real-time monitoring. Therefore, the research and clinical application of EVs biomarkers have become an international research hotspot. In order to encourage the development of screening and detection technologies for EVs biomarkers, effectively improve the accuracy of EVs biomarker diagnosis, and promote the transformation of EVs biomarker research results into the clinical application of disease, this review summarizes the significance of EVs in disease diagnosis and its biomarker screening strategy and clinical validation.