AbstractThe field of DNA nanotechnology has evolved beyond the realm of controllable movements and randomly shaped nanostructures, now encompassing a diverse array of nanomachines, each with unique nanostructures and biofunctional attributes. These DNA nanostructures boast exceptional characteristics such as programmability, integrability, biocompatibility, and universality. Among this variety, DNA walking nanomachines have emerged as one of the most prominent nanomotors, distinguished by their ingenious design and comprehensive functionality. In recent times, these DNA walkers have witnessed remarkable advancements in areas ranging from nanostructural designs to biological applications, including the creation of sophisticated biosensors capable of efficiently detecting tumor‐related biomarkers and bioactive substances. This review delves into the operational mechanisms of DNA walking nanomachines, which are driven by processes such as protease and DNAzyme action as well as strand displacement and photoactivated reactions. It further provides a comprehensive overview of DNA walking nanomachines with different dimensional (1D, 2D, and 3D) walking tracks. A subsequent section introduces the biosensing applications of DNA walking nanomachines including electrochemical, optical, and other biosensors. The review concludes with a forward‐looking perspective on the novel advancements and challenges in developing DNA walking nanomachine‐based biosensors.

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