Extracellular vesicles (EVs) are shed from most mammalian cells by different processes. EVs possess several distinct advantages, including excellent biocompatibility, good bio-stability and low immunogenicity. Moreover, they play significant roles in physiological and pathological processes. Challenges in EV research mainly concern highly efficient isolation, specific membrane surface engineering and further development of EV applications in biomedical fields. In this review, we summarize the recent and representative research regarding isolation, engineering and biomedical applications of EVs, which represent important research focus areas. These three aspects have not ever been systematically classified and summarized in previous reviews. Finally, we give our insights into the key issues concerning EVs and their future development for biomedical applications.