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Extracellular vesicles (EVs) are secreted by cells with a membrane structure and complex components such as DNA, RNA and proteins. These biomolecules play an important role in cell communication, cell proliferation, cell migration, vascularization, immune response and other physiological and pathological processes. Most current research on EVs focused on populations of EVs. Heterogeneity of EVs is neglected. Considering the heterogeneity of single EVs may offer critical molecular insights into cell-cell interactions, it is necessary to enhance our understanding about molecular characteristics from EVs derived from cell population to a single EV of derived from a single cell. This transformation is expected to provide a new insight into the understanding of cellular biology and the accurate description of the law of disease progress. In this article, we review the current research progress of single EV analysis technology for single EVs derived from cell population (SECP) and discuss its main applications in biological and clinical medicine research. After that, we propose the development direction, main difficulties and application prospect of single EV analysis technology for single EVs derived from single cells (SESC) according to our own research work, to provide new perspectives for the field of EV research., Competing Interests: Declaration of competing interest We declare that we have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this review paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)