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Extracellular vesicles (EVs) are lipid membrane enclosed nano-sized vesicles that are secreted by all known organisms. These vesicles are increasingly recognized as important circulating biomarkers for the diagnosis and prognosis of different diseases including various types of cancer, owing to their essential role in intercellular communication. EVs preserve heterogeneity in both physical properties and cargos, which makes it extraordinarily tough to fully exploit their clinical potential. Therefore, comprehensive characterization of single EVs and their sensitive detection are urgently demanded. In this article, we survey the latest progress in single EVs analysis with innovative discoveries in heterogeneity and highlight the various label-free and labelling approaches of single EVs detection. Furthermore, the state-of-the-art advances in single EV-detection based biomedical applications with especial emphasis on cancer investigations are summarized. To the end, the challenges and prospects for exploiting new system in the field of single EVs study are discussed. • A comprehensive review covering the recent progresses in single extracellular vesicle-based analysis. • Discussion on the significance of single extracellular vesicle analysis. • Focusing on the advanced analysis of single extracellular vesicles, especially in combination with microfluidic platform. • Review on the single extracellular vesicle analysis-based biomedical application with emphasis on cancer investigations. • Discussion on the existing obstacles and future perspectives on single extracellular vesicle research. [ABSTRACT FROM AUTHOR]