Extracellular vesicles (EVs) are the enigmatic messengers of intercellular communications and vital biological processes, including immune regulation, tissue regeneration, and disease progression. Despite their immense values as clinical diagnosis, the analysis of EVs poses significant challenges due to their small size and heterogeneity. This review focuses on the rapid evolution of fluorescence (FL) biosensing technologies. The convergence of FL biosensing technologies with advancements in biotechnology and nanotechnology has elaborated EV analysis, providing unprecedented sensitivity, specificity, and multiplexing capabilities. Here we overview three key areas of FL analyses of EVs: FL signal amplification technologies, Förster resonance energy transfer-based technologies, and innovative FL technologies. These cutting-edge methods, utilizing fluorescent dyes, enable precise visualization and simultaneous analysis of multiple EVs, offering insights into their diverse subpopulations and potential implications for biological processes, paving the way for novel diagnostic and therapeutic strategies.