Extracellular vesicles (EVs) are secreted by both eukaryotes and prokaryotes, and are present in all biological fluids of vertebrates, where they transfer DNA, RNA, proteins, lipids, and metabolites from donor to recipient cells in cell-to-cell communication. Some EV components can also indicate the type and biological status of their parent cells and serve as diagnostic targets for liquid biopsy. EVs can also natively carry or be modified to contain therapeutic agents (e.g., nucleic acids, proteins, polysaccharides, and small molecules) by physical, chemical, or bioengineering strategies. Due to their excellent biocompatibility and stability, EVs are ideal nanocarriers for bioactive ingredients to induce signal transduction, immunoregulation, or other therapeutic effects, which can be targeted to specific cell types. Herein, we review EV classification, intercellular communication, isolation, and characterization strategies as they apply to EV therapeutics. This review focuses on recent advances in EV applications as therapeutic carriers from in vitro research towards in vivo animal models and early clinical applications, using representative examples in the fields of cancer chemotherapeutic drug, cancer vaccine, infectious disease vaccines, regenerative medicine and gene therapy. Finally, we discuss current challenges for EV therapeutics and their future development.