Tumor　marker-responsive　drug　delivery　systems　have　been　developed　for　cancer　imaging　and　chemotherapy.　However,　improving　their　ability　of　controlled　drug　release　remains　a　challenge.　In　this　study,　we　have　developed　an　adenosine　triphosphate　(ATP)-responsive　DNA　nanohydrogel　for　specifically　activated　fluorescence　imaging　and　chemotherapy　in　cancer　cells.　Acrylamide　and　acrydite-modified　DNAs　were　polymerized　to　obtain　DNA-grafted　polyacrylamide　copolymers.　Then,　the　copolymers　acted　as　the　backbone　of　the　nanohydrogel　and　were　assembled　by　base　complementation　with　ATP　aptamer　linkers　to　construct　an　ATP-responsive　nanohydrogel.　Meanwhile,　the　chemotherapeutic　drug　doxorubicin　(DOX)　was　added　and　loaded　into　the　ATP-responsive　nanohydrogel　during　the　assembly　process.　After　endocytosis　by　cancer　cells　and　response　to　a　high　intracellular　ATP　level,　the　DOX-loaded　nanohydrogel　disassembled　due　to　the　formation　of　aptamer/ATP　complexes.　Subsequently,　the　released　DOX　played　a　role　in　fluorescence　imaging　and　chemotherapy　of　cancer　cells.　Through　the　ATP-responsive　property　and　satisfying　drug　delivery　capability,　this　nanohydrogel　realized　fluorescence　imaging　and　specific　cancer　cell　killing　capabilities　due　to　different　intracellular　ATP　levels　in　normal　and　cancer　cell　lines.　In　summary,　this　study　has　provided　a　novel　strategy　of　constructing　a　tumor　microenvironment-responsive　drug　delivery　system　triggered　by　the　tumor　markers　for　tumor　intracellular　imaging　and　chemotherapy.　©　2022　American　Chemical　Society.　All　rights　reserved.