Interactions between phenolics and protein play important roles in the stability and bioactivities of functional milks enriched in phenolic compounds. The interaction of rution and ferulic acid with casein were explored by spectroscopic analysis and antioxidant activity assay. Fluorescence spectroscopic analysis indicated that both rutin and ferulic acid could quench intrinsic fluorescence of casein through a static quenching procedure. The results of thermodynamic parameters demonstrated that the binding force of rutin and casein was mainly hydrophobic interaction, while hydrophobic interaction and hydrogen bond were the binding force between ferulic acid and casein. UV-Vis and synchronous fluorescence spectroscopy showed that the addition of rutin and ferulic acid affected the microenvironment around the tyrosine and tryptophan residues in casein, which caused the conformational change of casein. Analysis of fourier transform infrared spectroscopy and circular dichroism further indicated that the secondary structure of casein was changed, but not completely destroyed by rutin and ferulic acid.