Achieving　reliable　miRNA　detection　and　imaging　remains　a　critical　and　formidable　challenge.　To　address　this　challenge,　we　developed　a　three-dimensional　(3D)　DNA　walker　nanoprobe　(DWN),　derived　from　a　multifunctional　molecular　beacon　with　a　lipidic　moiety.　This　innovative　structure　employs　a　cholesterol-conjugated　multifunctional　molecular　beacon　(Chol-MMB)　assembled　through　hydrophobic　interactions,　thereby　bypassing　the　cumbersome　and　inefficient　process　of　immobilizing　DNA　strands　onto　rigid　surfaces.　Once　the　walker　nanoprobe　enters　cells,　the　binding　between　the　target　miRNA　and　the　nanoprobe　triggers　an　intermolecular　polymerization　process,　leading　to　a　conformational　change　in　DWN.　This　change　liberates　a　walking　strand,　initiating　the　walking　reaction.　As　a　result,　the　3D　DNA　walker　nanoprobe　illuminates　the　fluorophore,　enabling　precise　quantitative　analysis.　The　efficacy　of　the　DWN　was　demonstrated　by　the　limit　of　detection　of　0.326　fmol　for　miR-21　with　a　reaction　duration　of　1　h.　In　addition,　this　strategy　effectively　differentiates　various　cells　based　on　the　specific　miRNA.　Further　refinement　of　this　method　will　lead　to　an　efficacious　miRNA-based　platform　for　cell　typing　and　liquid　biopsy.　©　2024　Elsevier　B.V.