In this work, the binding of polyphenols isolated from coffee (PIC) with milk protein fractions was investigated by using different spectroscopic techniques as Fourier Transform Infrared Spectroscopic technique, high-performance liquid chromatography, and fluorescence quenching emission method. The polyphenols fraction was isolated from coffee powder using a chromatographic column packed with lipophilic Sephadex LH-20 gel, while there are different types of milk proteins skim milk 3% protein (SM), acid casein (beta-Casein (beta-CN), alpha-Casein (alpha-CN), kappa-Casein (kappa-CN)), and whey protein isolate (WPI) (beta-lactoglubulin (beta-Lg) and alpha-lactalbumin (alpha-La)) were investigated. From our study, the optimum pH for precipitating complex from the interaction of polyphenols with milk proteins, WPI, acid casein was 5, 8, and 7, respectively. Also, the fluorescence quenching method for polyphenols-proteins interaction exhibited fluorescence quenching in the descending order of alpha-CN > beta-Lg> SM3% > WPI > alpha-La > acid casein > beta-CN > kappa-CN for milk proteins. Furthermore, vibrational absorption mode of the functional groups confirmed of the appearing the polyphenols-proteins interaction. Besides, the percentage of interaction between milk protein fractions with PIC was mentioned using HPLC technique.