While　the　intracellular　imaging　of　miRNA　biomarkers　is　of　significant　importance　for　the　diagnosis　and　treatment　of　human　cancers,　DNA　assembled　nanoprobe　has　recently　attracted　considerable　attention　for　imaging　intracellular　biomolecules.　However,　the　complex　construction　process,　intrinsic　vulnerability　to　nuclease　degradation　and　the　limited　signal　transduction　efficiency　hamper　its　widespread　application.　In　this　contribution,　based　on　persistent　autonomous　molecular　motion　of　DNAzyme　walker　along　a　nano-substrate　track,　a　DNA　nanosphere　probe　(PNLD)　is　developed　for　the　sensitive　intracellular　miR-21　imaging.　Specifically,　DNA　nanosphere　(called　PN,　single-molecule　nano-track)　is　assembled　from　only　one　palindromic　substrate,　into　which　the　locking　strand-silenced　DNAzymes　(LD)　are　installed　in　a　controlled　manner.　PNLD　(made　of　PN　and　LD)　can　protect　all　DNA　components　against　nuclease　attack　and　maintain　its　structural　integrity　in　serum　solution　over　24　h.　Upon　the　activation　by　target　miRNA,　DNAzyme　walker　can　move　on　the　substrate　scattered　within　PNLD　(or　on　the　surface)　and　between　different　PNLD　objects　and　cleave　many　DNA　substrates,　generating　an　amplified　signal.　As　a　result,　miR-21　can　be　detected　down　to　6.83　pM　without　the　detectable　interference　from　co-existing　nontarget　miRNAs.　Moreover,　PNLD　system　can　accurately　screen　the　different　expression　levels　of　miR-21　within　the　same　type　of　cells　and　different　types　of　cells,　which　is　consistent　with　gold　standard　polymerase　chain　reaction　(PCR)　assay.　Via　changing　the　target　recognition　sequence,　the　PNLD　system　can　be　suitable　for　the　intracellular　imaging　of　miR-155,　exhibiting　the　desirable　universality.　In　addition,　the　DNAzyme　walker-based　PNLD　system　can　be　used　to　distinguish　cancer　cells　from　healthy　cells,　implying　the　potential　application　in　cancer　diagnosis　and　prognosis.　©　2024　Elsevier　B.V.