DNA-based nanomachines have received increasing attention due to their great potential to mimic natural biological motors and create novel modes of motion. Here, we report a DNAzyme-based walking machine, which can operate in living cells after triggered by intracellular miRNA-21. The walking machine is constructed by assembling DNAzyme walking strands and FAM-labeled substrate strands on a single gold nanoparticle (AuNP). The DNAzyme walking strand is first silenced by a blocker strand. After cellular uptake, DNAzyme-based walker can be triggered by intracellular miRNA-21 and autonomously walk along the AuNP-based 3D track fueled by DNAzyme-catalyzed substrate cleavage. Each walking step results in the cleavage of a substrate strand and the release of a FAM-labeled DNA strand, allowing real-time monitoring of the operation of the machine. The DNAzyme-based walking machine has been successfully applied to image and monitor miRNA-21 expression levels in living cells with excellent specificity and reliability. This walking machine would hold great potential in the miRNA associated biological research and disease diagnostics.