DNA-templated copper nanoparticles (CuNPs) have recently received considerable interest as functional fluorescent probes for biochemical analysis. In this work, a novel ATP-dependent ligation reactions (ATP-DLR) based ATP assay strategy was proposed by using hairpin-shaped (HS) DNA templated CuNPs as a fluorescent probe. Nick sealing by T4 DNA ligase leads to the formation of intact HS DNA, which can resist the exonuclease cleavage and be taken as the template for CuNPs formation, resulting in strong fluorescence. The proposed ATP detection is label free, sensitive and highly selective, and it has good linearity from 0.02 to 4 μM and a detection limit of 7 nM. This strategy is expected to promote the exploitation and application of DNA-templated CuNPs in biochemical and biomedical studies, and holds great promise in fluorescence detection for other ligation-related biomolecules.