Abstract: With the rapid development of DNA nanotechnology, DNA molecules with various structures and functions are used to construct programmable nanomachines with dynamic behaviors. Thereinto, DNA walker possesses the advantages of the cascade signal amplification generated by the autonomous movement along the predetermined track by driving force, which is widely applied in the biosensing, biological imaging, and drug delivery fields, thus showing the great application potential for rapid and ultrasensitive detection of contaminants in food. This review first briefly introduces the basic principles of DNA walker, and the strategies for improving the efficiency of DNA walker was described in detail, then summarizes its applications in the food safety detection. Finally, the future research directions of DNA walkers are prospected to provide theoretical guidance for its real applications.