A label-free aptasensor has been fabricated in order to detect adenosine triphosphate (ATP) using turn-on fluorescence DNA-Ag NCs. The fluorescence of the DNA-Ag NCs could increase remarkably with the addition of ATP mainly because ATP specifically interacts with its aptamer to change the microenvironment of the darkish DNA-Ag NCs located at one terminus or two termini due to the conformational alteration of the aptamer structure. The proposed sensor can detect ATP in a linear range of 6–27 mM with a good detection limit of 5.0 μM. Additionally, the proposed method succeeded in detecting ATP in fetal bovine serum.