In this work, the binding of caffeine (CAF) with nicotinamide (NIC) has been investigated by means of fluorescence quenching and UV-Vis spectroscopic techniques. The results showed that CAF could effectively quench the intrinsic fluorescence of NIC due to a static quenching mechanism rather than a dynamic one. The key parameters of the process: the quenching constant and the bimolecular quenching rate one, the number of binding sites (n ≈ 1), as well as the thermodynamic properties of NIC with CAF were calculated. The binding constants were 6.500 and 5.577 kL/mole at 295 and 303 K respectively. The thermodynamic parameter values determined using the Van’t Hoff’s equation (ΔH = –14.220 kJ/mole, ΔS = 22.764 J/(mole∙K)) indicated that the binding process was continuous and electrostatic forces had a major role in the reaction of CAF with NIC molecules. Similarly, the UV-Vis absorption spectra of the interaction were studied and used to confirm the fluorescence quenching mechanism of the molecules.