Although　DNA　probes　have　attracted　increasing　interest　for　precise　tumor　cell　identification　by　imaging　intracellular　biomarkers,　the　requirement　of　commercial　transfection　reagents,　limited　targeting　ligands,　and/or　non-biocompatible　inorganic　nanostructures　has　hampered　the　clinic　translation.　To　circumvent　these　shortcomings,　a　reconfigurable　ES-NC　(Na+-dependent　DNAzyme　(E)-based　substrate　(S)　cleavage　core/shell　DNA　nanocluster　(NC))　entirely　from　DNA　strands　is　assembled　for　precise　imaging　of　cancerous　cells　in　a　successive　dual-stimuli-responsive　manner.　This　nanoprobe　is　composed　of　a　strung　DNA　tetrahedral　satellites-based　protective　(DTP)　shell,　parallelly　aligned　target-responsive　sensing　(PTS)　interlayer,　and　hydrophobic　cholesterol-packed　innermost　layer　(HCI　core).　Tetrahedral　axial　rotation-activated　reconfiguration　of　DTP　shell　promotes　the　exposure　of　interior　hydrophobic　moieties,　enabling　cholesterol-mediated　cellular　internalization　without　auxiliary　elements.　Within　cells,　over-expressed　glutathione　triggers　the　disassembly　of　the　DTP　protective　shell　(first　stimulus),　facilitating　target-stimulated　signal　transduction/amplification　process　(second　stimuli).　Target　miRNA-21　is　detected　down　to　10.6　fM　without　interference　from　coexisting　miRNAs.　Compared　with　transfection　reagent-mediated　counterpart,　ES-NC　displays　a　higher　imaging　ability,　resists　nuclease　degradation,　and　has　no　detectable　damage　to　healthy　cells.　The　blind test　demonstrates　that　the　ES-NC　is　suitable　for　the　identification　of　cancerous　cells　from　healthy　cells,　indicating　a　promising　tool　for　early　diagnosis　and　prediction　of　cancer.　©　2024　Wiley-VCH　GmbH.