Accurate　detection　and　imaging　of　low-abundance　microRNA　(miRNA)　in　living　cells　are　essential　for　the　diagnosis　and　prognosis　of　diseases.　Designing　nanoprobes　with　resistance　to　enzyme　degradation,　effective　cell-binding,　and　efficient　signal　amplification　is　crucial　for　in　vivo　imaging.　In　this　study,　we　present　an　aptamer-tethered　DNA　origami　amplifier　(ADOA)　that　functions　inside　living　cells　to　detect　miRNA　with　high　sensitivity　and　stability.　In　the　design,　cancer　cell-targeting　aptamers　were　tethered　onto　the　border　of　the　DNA　origami　to　improve　the　discrimination　between　cancer　cells　and　normal　cells.　Two　substrate　modules　for　the　intramolecular　entropy-driven　reaction　(EDR)　circuit　were　alternately　arranged　on　the　DNA　origami　plane.　The　target　miRNA　will　initiate　the　sequential　hybridization　of　the　two　substrate　modules　on　the　DNA　origami,　generating　amplified　fluorescence　signals.　The　proposed　ADOA　achieved　an　accelerated　cascade　reaction　due　to　the　＇confinement　effect＇and　significantly　enhanced　the　sensitivity　compared　with　a　traditional　EDR.　Meanwhile,　with　the　rigid　structure　of　the　DNA　origami,　the　ADOA　possessed　excellent　signalling　stability　in　living　cells.　Therefore,　the　ADOA　could　expand　the　application　of　DNA　origami　in　miRNA　sensing　and　has　potential　value　in　early-stage　clinical　diagnosis.　This　journal　is　©　The　Royal　Society　of　Chemistry.