Extracellular vesicles (EVs) are nanosized vesicles enclosed in a lipid membrane that are sustainably released by nearly all cell types. EVs have been deemed as valuable biomarkers for diagnostics and effective drug carriers, owing to the physiological function of transporting biomolecules for intercellular communication. To investigate their biological properties, efficient labeling strategies have been constructed for EV research, among which fluorescence labeling exerts a powerful function due to the capability of visualizing the nanovesicles with high sensitivity both in vitro and in vivo. In one aspect, with the help of functional fluorescence tags, EVs could be differentiated and categorized in vitro by various analytical techniques, which exert vital roles in disease diagnosis, prognosis, and treatment monitoring. Additionally, innovative EV reporters have been utilized for visualizing EVs, in combination with powerful microscopy techniques, which provide potential tools for investigating the dynamic events of EV release and intercellular communication in suitable animal models. In this feature article, we survey the latest advances regarding EV fluorescence labeling strategies and their application in biomedical application and in vivo biology investigation, highlighting the progresses in individual EV imaging. Finally, the challenges and future perspectives in unravelling EV physiological properties and further biomedical application are discussed.