In recent years, research has increasingly focused on the biogenesis of extracellular vesicles (EVs) and the sorting mechanisms for their contents. Mitochondria can be selectively loaded into EVs, serving as a way to maintain cellular mitochondrial homeostasis. EV-mediated mitochondrial transfer has also been shown to greatly impact the function of target cells. Based on the mechanism of EV-mediated mitochondrial transfer, therapies can be developed to treat human diseases. This review summarizes the recent advances in the biogenesis and molecular composition of EVs. It also highlights the sorting and trafficking mechanisms of mitochondrial components into EVs. Furthermore, it explores the current role of EV-mediated mitochondrial transfer in the development of human diseases, as well as its diagnostic and therapeutic applications.