The detailed characterizations of the binding interactions between food additive tartrazine (TZ) and beta-lactoglobulin (beta-LG) have been investigated through spectroscopic techniques combined with a molecular modeling study. A series of analyses, such as hyperchromic change in the UV-visible spectra, temperature-dependent quenching constant, time-resolved fluorescence, and Rayleigh scattering measurements, show that quenching of beta-LG proceeds by a static quenching mechanism. TZ specifically binds with beta-LG in a stoichiometry ratio of 1:1, and the observed binding constants (10(4), K) are 7.64, 9.13, 9.72, and 10.79 at 293, 298, 303, and 308 K, respectively. However, the curious results of binding constants (K) with temperature, encountered in the static quenching, have been well explained on the basis of Le Chatelier's principle. Thermodynamic data and pH-dependent studies along with the surface hydrophobicity binding displacement assay reveal that the durable mode of binding is chiefly entropy-driven, revealing noteworthy interactions of such ionic molecules with the hydrophobic part of beta-LG. The modulation of protein conformation has been investigated through steady-state absorption spectroscopy, synchronous emission spectroscopy, circular dichroism, and dynamic light scattering studies. TZ acts as a potential inhibitor in fibrillogenesis. Furthermore, the molecular docking study offers accurate insights about the binding of TZ with beta-LG, in consistence with the experimental results. This study would be helpful in pharmaceutical, food, and industrial engineering chemistry research.