The inclusion of acridinedione dyes (ADR) with β-cyclodextrins (β-CD) in the presence of 0.6% methanol is studied using absorption and fluorescence techniques. The association constant was determined by using the Benesi−Hildebrand plot and also by nonlinear regression analysis. A stoichiometry of 1:1 was found for β-CD/ADR (3 − 5). The nonobservation of the changes in the absorption or the fluorescence in the case of dyes 1 and 2 clearly indicates that the phenyl substituent at the −N atom in the ADR (3 − 5) is included in the β-CD cavity. Thermodynamic parameters ΔH and ΔS values obtained from the temperature-dependent binding constants of the β-CD/ADR (3 − 5) system are reported. In the presence of β-CD, the fluorescence of ADR (4) dye shows single exponential decay, consistent with the completion of complex formation, and ADR (3, 5) showed a double exponential decay, consistent with the equilibrium between free and complexed forms. The binding constants and thermodynamic factors are systematically resolved through lifetime analysis.