The ability to design nanostructures with arbitrary shapes and controllable motions has made DNA nanomaterials used widely to construct diverse nanomachines with various structures and functions. The DNA nanostructures exhibit excellent properties, including programmability, stability, biocompatibility, and can be modified with different functional groups. Among these nanoscale architectures, DNA walker is one of the most popular nanodevices with ingenious design and flexible function. In the past several years, DNA walkers have made amazing progress ranging from structural design to biological applications including constructing biosensors for the detection of cancer‐associated biomarkers. In this review, the key driving forces of DNA walkers are first summarized. Then, the DNA walkers with different numbers of legs are introduced. Furthermore, the biosensing applications of DNA walkers including the detection‐ of nucleic acids, proteins, ions, and bacteria are summarized. Finally, the new frontiers and opportunities for developing DNA walker‐based biosensors are discussed.