Small extracellular vesicle (sEV) also known as exosome carries of intercellular information and genetic material transfer, e.g., DNA, RNA, proteins, lipids, and other active substances. However, conventional detection techniques do not meet the requirements of sEVs analysis. Recently, electrochemiluminescence (ECL) biosensors have become powerful tools in the detection of sEVs owing to their attractive properties with identification strategies and unique potential- and spatial-controlled ability. Therefore, in this review, the latest advances and principal issues in the ECL biosensors for sEV detection are systematically discussed. Especially, the sensing mechanisms and signal transduction strategies involved in sEV identification are discussed in detail to provide insights into the design and construction of ECL biosensors, such as DNA logical computation, proximity labeling, proximity ligation assay, mediated-membrane fusion strategy, lab-on-a-chip systems, as well as artificial intelligence. Subsequently, the recent application of ECL biosensors for clinic sEV diagnosis is further emphatically introduced, including tumorous sEV identification, peritoneal metastasis diagnosis, tumor microenvironment monitoring, phenotypic profiling, and asthma evaluation. Finally, the potential challenges and future directions of sEV sensing systems are highlighted. It provides a distinct perspective for researchers to construct ECL biosensors and enlightens the fast development of sEV detection in biological research, clinical use, and commercialization.