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.