Dynamic　DNA　self-assembly　(DDSA)　has　been　developed　for　highly　sensitive　monitoring　intracellular　cancer　biomarkers　via　in　situ　signal　amplification.　However,　each　step　of　reaction　depends　on　the　diffusion　of　DNA　strands　within　the　three-dimensional　cellular　environment,　which　greatly　limits　the　reaction　efficiency.　Herein,　we　proposed　a　localized　catalytic　hairpin　assembly　(CHA)　reaction　on　DNA　Origami　with　the　purpose　of　detection　and　imaging　of　intracellular　microRNA-21.　In　this　strategy,　8　pairs　of　DNA　hairpins　of　CHA　reaction　alternately　hybridized　to　the　protruding　single　strands　of　DNA　origami　rectangle,　so　that　the　whole　reaction　process　is　constrained　in　a　compact　space　and　independent　on　the　diffusion　of　the　DNA　strands.　Obviously,　this　localized　CHA　reaction　offered　faster　reaction　kinetics.　Our　results　confirmed　that,　compared　to　the　traditional　CHA　reaction,　constraining　DNA　hairpins　onto　a　rectangular　DNA　origami　increased　the　reaction　rate　and　the　sensitivity.　In　addition,　DNA　origami　rectangle　could　also　play　as　a　carrier　to　deliver　DNA　hairpins　to　the　cytoplasm　and　protect　DNA　hairpins　from　the　attack　of　nuclease.　This　strategy　provided　a　versatile　platform　which　could　be　easily　integrated　with　reported　DDSA-based　strategy　and　therefore　showing　a　wide　application　prospect.　©　2021