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The interaction of natural polyphenolic compounds (rutin) with β-lactoglobulin (BLG) was carried out by using several optical spectroscopic (UV–visible spectroscopy, fluorescence quenching measurements, synchronous fluorescence, 3D fluorescence spectroscopy and far-UV CD measurements), molecular docking and molecular dynamics (MD) simulation methods. The fluorescence quenching results confirmed that fluorescence intensity of BLG is quenched by rutin and the quenching constant is increased with increase in temperature. The quenching mechanism between rutin-BLG was found to be dynamic in nature. The thermodynamic parameter particularly ΔH⁰ and ΔS⁰ obtained through fluorescence measurements clearly indicated that hydrophobic forces are majorly involved in the rutin-BLG interaction. The UV-absorption, synchronous and three-dimensional fluorescence results displayed that the micro …