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With the continuous development of DNA nanotechnology, researchers have focused increasing attention on the use of DNA molecules with diverse structural and functional properties to construct nanomachines. Among the various molecular machines, DNA walker is one of the most sophisticated DNA devices either in design or construction. Its key point is to drive the walking strand to move steadily and progressively along the tracks by the motor, and further to implement the generation and amplification of signals, cargo delivery, molecular computing and so on. This review provides a systematic and comprehensive overview of the design principles for DNA walkers with typical examples and in-depth discussion, as well as an exhaustive description of its application in the field of biosensors. Meanwhile, it also summarizes the performance advantages and challenges of DNA walker biosensors, thus offering a …