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Abstract Extracellular vesicles (EVs) exhibiting versatile biological functions provide promising prospects as natural therapeutic agents and drug delivery vehicles. For future clinical translation, revealing the fate of EVs in vivo, especially their accumulation at lesion sites, is very important. The continuous development of in vivo imaging technology has made it possible to track the real‐time distribution of EVs. This article reviews the applications of mammal‐, plant‐, and bacteria‐derived EVs in tumor therapy, the labeling methods of EVs for in vivo imaging, the advantages and disadvantages of different imaging techniques, and possible improvements for future work.