With the continuous development of DNA nanotechnology, DNA walkers have attracted increased attention because of their autonomous and progressive walking along predesigned tracks. Compared with the traditional DNA walkers, the emerged multipedal DNA walkers showed their special charm with sustainable walking capability, higher reaction efficiency, expanded walking region, and improved amplification capability. Consequently, multipedal DNA walkers have developed rapidly and shown potential in biosensing applications. Hence, in this review, we make a comprehensive representation of the engineering strategy of multipedal DNA walkers, which focused on the design of multiple walking strands as well as the construction of tracks and driving forces. Meanwhile, the application of multipedal DNA walkers in biosensors has been thoroughly described according to the type of biosensing signal readout. By illustrating some representative works, we also summarized the merits and challenges of multipedal DNA walker-based biosensors and offered a deep discussion of the latest progress and future.