DNA-based nanoprobes have attracted extensive interest in the field of bioanalysis. Notably, engineered DNA nanoprobes that can respond to multiple pathological parameters are desirable to detect targets precisely. Here we design a split aptamer/DNAzyme (aptazyme)-based DNA probe for fluorescence detection of ATP and further develop a cooperatively activatable DNA nanoprobe for tumor-specific imaging of ATP in vivo. The DNA nanoprobes comprising split aptazyme-coated MnO2 nanovectors have high stability and are synergistically activated by multiple biomarkers, GSH and ATP. Upon stimuli by overexpressed GSH in tumor cells, this DNA nanoprobe can release the aptazyme and self-supply cofactor Mn2+ of the DNAzyme. Sequentially, intracellular ATP induces the proper folding of the split ATP aptamer and Mn2+-dependent DNAzyme, which activates the specific cleavage of substrate and generates the optical readout signal. This nanoprobe exhibits remarkable resistance to enzymatic degradation, satisfactory biosafety, identifies ATP specifically within cancer cells, and selectively lights up solid tumors. Our research provides a reliable method for ATP imaging in cancer cells and opens a new avenue for biochemical research and highly accurate disease diagnosis.