Nucleic acids are regarded as reliable biomarkers for the early diagnosis of various diseases. By ingeniously combining a transduction hairpin (THP) with the toehold-mediated strand displacement reaction (TSDR) to form three-leg DNAzyme walkers, for the first time, we constructed a label-free and sensitive electrochemical sensing system for the amplification detection of target nucleic acids. With microRNA-155 (miR-155) as a model target, the feasibility of the biosensing strategy and the conformational states of DNA in the recognition process were studied in detail on the basis of electrochemical and dual polarization interferometry techniques. With the assistance of THP, miR-155 indirectly triggered the TSDR between three hairpins (H1, H2, and H3), then massive Mg2+-dependent three-leg DNAzyme walkers were formed in aqueous solutions. After the binding/cleaving/moving process of three-leg DNAzyme walkers on the electrode surface modified with substrate hairpins (SHPs), a number of single-stranded DNAs (ssDNAs) were generated. Hence, the interaction of methylene blue (MB) with the duplex section of SHPs was impeded, which brought about a decreased electrochemical signal. Benefiting from the cyclic amplification of the TSDR and the higher cleavage activity of three-leg DNAzyme walkers, the proposed sensing strategy showed remarkable improvement in sensitivity with a low detection limit of 0.27 fM for miR-155. Owing to the precise design of the THP, this method exhibited excellent specificity to distinguish miR-155 from the single-base and triplex-base mismatched sequences. This sensing strategy importing the flexible THP can be utilized to detect various nucleic acid biomarkers by only redesigning the THP without changing the main circuit or reporter constructs, showing the great versatility and potential for the early diagnostics and biological analysis.