In　situ　monitoring　microRNA　(miRNA)　expression　in　vivo　holds　immense　potential　for　directly　visualizing　the　occurrence　and　progression　of　tumors.　However,　the　significant　barrier　to　developing　a　probe　that　can　overcome　the　low　abundance　of　miRNAs　while　providing　an　output　signal　with　unlimited　tissue　penetration　depth　remains　formidable.　In　this　study,　we　developed　a　DNA　machine-based　magnetic　resonance　imaging　nanoprobe　(MRINP)　for　amplified　detection　of　miR-21　in　vivo.　The　MRINP　was　constructed　with　superparamagnetic　Fe3O4　nanoparticles　(NPs),　paramagnetic　Gd-DOTA　complexes,　and　miR-21-activated　DNA　machines;　the　DNA　machine　was　composed　of　hairpin　DNAzyme　(HD)　strands　serving　as　the　DNAzyme　walker　and　hairpin　substrate　(HS)　strands　serving　as　the　track.　Once　uptake　into　tumor　cells,　the　intracellular　miR-21　specifically　recognized　and　hybridized　with　the　HD　strand,　restoring　the　activity　of　DNAzyme.　Subsequently,　the　DNAzyme　walker　autonomously　traveled　on　the　surface　of　MRINP,　and　each　step　movement　of　the　DNAzyme　walker　resulted　in　the　cleavage　of　its　substrate　strands　and　the　ensued　release　of　the　Gd-DOTA　complex-labeled　oligonucleotides,　turning　on　the　T1　signal　of　Gd-DOTA　complexes　for　in　situ　imaging　of　miR-21　in　tumor-bearing　mice.　This　strategy　would　offer　a　promising　approach　for　mapping　tumor-specific　biomarkers　in　vivo　with　unlimited　penetration　depth.　©　2024　Elsevier　B.V.