In this work, a novel strategy for preparation of bipedal DNA walker (BDW) based on hybridization chain reaction (HCR) with the assistance of Exonuclease III (Exo III) was proposed. Based on this strategy, an electrochemical biosensor was constructed to achieve sensitive detection of CYFRA 21-1 DNA. Firstly, target recognition and circulation were achieved through a one-step catalytic hairpin assembly (CHA) reaction. For further amplification, hybridization chain reaction (HCR) was employed to form duplex-stranded DNA (dsDNA) nanostructure in homogeneous solution. In particular, the elongated single strand of the hairpin DNA for HCR was designed as the Mg²⁺ DNAzyme sequence. With the assistance of Exo III, dsDNA nanostructure can be digested and transformed into large amounts of BDW. These BDW can cleave the signal probe driven by Mg²⁺, which was modified on the electrode surface and thus achieved “signal-off” detection of target. This BDW preparation method based on HCR with the digestion of Exo III converted one target input into large amount of BDW. Coupled with the walking cleavage of BDW, a series of cascade amplification endowed high sensitivity with this biosensor and realized ultrasensitive detection of target DNA with the detection limit as low as 3.01 aM.