Adenosine 5′-triphosphate (ATP) is a functional molecule associated with many important biological processes. Fluorescent detection methods for ATP with facile performance and high selectivity are in demand. One of the possible multi-membered arrays assembled between DHBO and Ga3+ ions was conducted in aqueous solution, which can selectively recognize ATP with fluorescence enhancement from ADP, AMP and other structurally similar nucleoside triphosphates in vitro and in vivo. ATP facilitates the interaction between DHBO and Ga3+ ions, resulting in the fluorescence increase. The detection limit for ATP was calculated to be 5.49×10−7 M, which is much lower than that of intracellular concentrations (1–10 mM). In addition, DHBO–Ga3+ can be applied to detect ATP-relevant enzyme activity.