Extracellular vesicles (EVs), as natural carriers, are regarded as a new star in nanomedicine due to their excellent biocompatibility, fascinating physicochemical properties, and unique biological regulatory functions. However, there are still some challenges to using natural EVs, including poor targeting ability and the clearance from circulation, which may limit their further development and clinical use. Nucleic acid has the functions of programmability, targeting, gene therapy, and immune regulation. Owing to the engineering design and modification by integrating functional nucleic acid, EVs offer excellent performances as a therapeutic system . This review briefly introduces the function and mechanism of nucleic acid in the diagnosis and treatment of diseases. Then, the strategies of nucleic acid-functionalized EVs are summarized and the latest progress of nucleic acid-functionalized EVs in nanomedicine is highlighted. Finally, the challenges and prospects of nucleic acid-functionalized EVs as a promising diagnostic system are proposed.