A new and sensitive spectrofluorimetric method has been proposed to determine trace amount of adenosine disodium triphosphate (ATP). The method is based on the fluorimetric interaction between gatifloxacin (GFLX)–aluminium (III) (Al³⁺) complex and ATP and studied using UV‐visible and fluorescence spectroscopy. Weak luminescence spectra of Al³⁺ were enhanced after complexation with GFLX at 423 nm upon excitation at 272 nm due to energy transfer from the ligand to the Al³⁺ ion. It was observed that the FL emission spectrum of GFLX–Al³⁺ was enhanced significantly by the addition of ATP. Under the optimal conditions, the enhancement of FL intensity at 423 nm was responded linearly with the concentration of ATP in the range 1.3 × 10^–10 – 1.0 × 10^–8 mol L^–1 with correlation coefficient (r) of 0.9981. The limit of detection (LOD) was found to be 1.1 × 10^–11 mol L^–1 for ATP with the standard deviation (RSD) of 1.21% for five repeated measurement of 2.3 × 10^–8 mol L^–1 ATP. The presented method is simple, sensitive, free from coexisting interferents and can be applied successfully to determine ATP in the real samples. Copyright © 2015 John Wiley & Sons, Ltd.