Electrochemical biosensors are characterized by rapid response, miniaturization, portability, and ease of operation. With tunable nanostructure, DNA has been comprehensively combined with electrochemical devices to design high-sensitivity and selectivity biosensors for the achievement of disease diagnosis, food safety, and environmental monitoring. In recent years, DNA-functionalized electrochemical biosensors have made significant research progress. In this article, the recent research progress of DNA-functionalized electrochemical biosensors for in vitro and in vivo disease diagnosis was reviewed. The structure and sensing principles of DNA-functionalized electrochemical biosensors were first summarized. The preparation of DNA-functionalized electrochemical biosensors based on nanomaterials was introduced in detail. Meanwhile, the latest evolution of integrated and portable DNA-functionalized electrochemical biosensors for in vitro disease diagnosis was summarized. For a further step, the construction of implantable DNA-functionalized electrochemical biosensors for in vivo and real-time disease monitoring was overviewed. Finally, the challenges and outlook of DNA-functionalized electrochemical biosensors were discussed to provide a guideline for the future development of DNA-functionalized electrochemical biosensors.