Fluorescence self-quenching occurs at high concentration. Inhibition of self-quenching by inclusion of fluorescence emitters inside the hydrophobic cavity of β-cyclodextrin (β-CD) has been addressed taking the example of the fluorescence behavior of Tramadol hydrochloride. Indeed complexation by β-CD enhanced fluorescence emission of Tramadol under conditions where self-quenching was operative. A quantitative account of self-quenching and its inhibition by β-CD was done through determination of complexation equilibrium by 1H NMR experiments and a detailed study of absorption and fluorescence properties. Tramadol and β-CD associate as a complex of 1:1 stoichiometry with a formation constant K11 = 260. Complexation of Tramadol by β-CD does not cause modification of its absorbance and fluorescence spectra. Fluorescence self-quenching of Tramadol above ∼ 1 mmol·L−1 was characterized by a Stern–Volmer constant K = 810 L·mol−1. Inhibition of self-quenching by formation of an inclusion complex was manifested by lower Stern–Volmer constants in the presence of β-CD. Such study required a correct account of Inner Filter Effects on fluorescence, which is mandatory in all physicochemical studies using fluorescence where concentrations are rather high.