A new spectrofluorimetric method is developed for determination of adenosine disodium triphosphate (ATP). The interactions between prulifloxacin (PUFX)-Tb(3+) complex and adenosine disodium triphosphate has been studied by using UV-vis absorption and fluorescence spectra. Using prulifloxacin-Tb(3+) as a fluorescence probe, under the optimum conditions, ATP can remarkably enhance the fluorescence intensity of the prulifloxacin-Tb(3+) complex at lambda=545 nm and the enhanced fluorescence intensity is in proportion to the concentration of ATP. Optimum conditions for the determination of ATP were also investigated. The dynamic range for the determination of ATP is 4.0 x 10(-7) to 2.0 x 10(-5) mol L(-1), and the detection limit (3 sigma/k) is 1.7 x 10(-8) mol L(-1). This method is simple, practical and relatively free interference from coexisting substances and can be successfully applied to determination of ATP in real pharmaceutical samples. The mechanism of fluorescence enhancement of prulifloxacin-Tb(3+) complex by ATP was also discussed.