Extracellular vesicles (EVs) are critical mediators of intercellular communication, carrying bioactive cargo and displaying diverse surface components that reflect their cellular origins and functions. The EV surface, composed of proteins, lipids, and glycocalyx elements, plays a pivotal role in targeting recipient cells, mediating biological interactions, and enabling selective cargo delivery. This review comprehensively examined the molecular architecture of EV surfaces, linking their biogenesis to functional diversity, and highlights their therapeutic and diagnostic potential in diseases such as cancer and cardiovascular disorders. Additionally, we explore emerging applications of EVs, including machine-learning-assisted analysis, chemical integration, and cross-system combinations. The review also discusses some key challenges in the clinical translation of EV-related technologies.