Extracellular vesicles (EVs) play a crucial role in intercellular communication, participating in various physiological and pathological processes, and showing great application potential in disease diagnosis, therapy and prognosis. Nevertheless, current EVs isolation and detection technologies are confronted with numerous challenges, such as the dilemma of balancing isolation efficiency and purity, the complex and time-consuming operation process, and the limited detection sensitivity. This paper reviews the current research status of EVs isolation and detection methods, analyzes the advantages and limitations of traditional techniques like ultracentrifugation, ultrafiltration and polymer precipitation, and explores the development prospects of emerging technologies such as microfluidic technology and single-vesicle detection. The article emphasizes the impact of EVs heterogeneity on isolation and detection, and explains the potential of emerging technologies in overcoming the defects of traditional methods. In addition, it delves into the clinical translation research progress of EVs in fields like oncology and tissue repair, points out key barriers such as the lack of technical standardization and insufficient clinical validation, and proposes future research directions to promote the basic research and clinical translation of EVs. Overall, this study aims to provide theoretical and technological innovation support for EVs-related research and clinical applications.