A　stringent　DNA　probe　to　profile　microRNA　(miRNA)　expression　within　a　specific　cell　remains　a　key　challenge　in　biology.　To　address　this　issue,　an　intracellular　ATP-activated　Y-DNA　probe　for　accurate　imaging　of　miRNA　in　living　cells　was　designed.　Y-DNA　was　based　on　the　fabrication　of　tripartite　function　modules,　which　consisted　of　a　folate　(FA)-modified　targeting　module,　an　ATP　aptamer-sealed　driver,　and　a　miRNA　sensing　module.　The　Y-DNA　probe　could　be　specifically　activated　by　ATP　after　it　efficiently　internalized　into　FA-receptor-overexpressed　cells　based　on　caveolar-mediated　endocytosis,　leading　to　the　activation　of　the　miRNA　sensing　module.　The　activated　Y-DNA　probe　allowed　for　the　imaging　of　miRNA　in　living　cells　with　high　sensitivity.　The　design　of　the　ATP-activated　Y-DNA　sensor　opens　the　door　for　bioorthogonal　miRNA　imaging　and　promotes　the　development　of　various　responsive　DNA　molecular　probes　with　enhanced　anti-interference　ability　for　clinical　diagnosis.　©　2023　by　the　authors.