We　report　a　tetrahedron-based　DNAzyme　probe　(Tetra-ES)　for　intracellular　miRNA　detection.　Two　DNA　tetrahedra　(Tetra)　were　arranged　at　the　different　positions　of　the　enzyme　(E)/substrate　(S)　complex　in　a　unique　direction.　A　Na+-dependent　DNAzme　was　designed　to　be　initially　locked　to　inhibit　the　activity　of　the　DNAzyme.　Fluorescence　imaging　and　gel　electrophoresis　analyses　demonstrated　that　the　silenced　DNAzyme　could　be　specifically　initiated　by　intracellular　target　miRNA.　The　activated　DNAzyme　repeatedly　cleaved　the　substrates,　allowing　a　controllable　signal　transduction　and　amplification　effect.　The　combination　of　spatially　controlled　arrangement　of　DNA　tetrahedra　with　the　stimuli-responsive　behavior　of　the　locked　DNAzyme　improved　cell　permeability　and　desirable　nuclease　resistance.　The　Tetra-ES　detector　exhibited　at　least　10　times　higher　detection　sensitivity　(LOD　of　16　pM)　than　that　of　the　nonamplification　molecular　beacon　counterpart　and　was　capable　of　discriminating　the　miRNA　target　from　the　corresponding　family　members.　The　expression　levels　of　target　miRNA　inside　the　cells　of　interest　as　well　as　different　miRNAs　inside　the　same　type　of　cell　lines　were　reliably　screened　utilizing　the　Tetra-ES　detector.　As　an　intracellular　probe,　Tetra-ES　may　provide　valuable　insight　into　developing　a　homogeneous　DNA　nanostructure-based　controllable　signal　transduction　strategy　suitable　for　detection　of　miRNA　and　potential　application　to　cancer　diagnosis,　prognosis,　and　therapeutics.　Copyright　©　2019　American　Chemical　Society.