The binding of rhaponticin to bovine serum albumin (BSA)-bovine lactoferrin (BLF) and the factors that affect BSA-BLF interaction have been studied by fluorescence spectroscopy and Fourier transform infrared spectroscopy. In the fluorescence experiment, RT quenched the fluorescence intensity of mixed proteome and the maximum emission wavelength of BSA, BLF and BSA-BLF proteins system. RT caused obvious red-shift fluorescence for an interaction between RT and proteome. The interaction between RT and proteome was impacted by single-component protein molecular interactions and the interaction between RT-BSA and RT-BLF, the microenvironment of solutions were the factors impacting the interactions between RT and proteome, which impacted quantitative expression of the general environment micro environmental factors. In the Fourier transform infrared spectroscopy, the secondary conformation of protein molecules of single component in the protein group were changed, and the difference of the molecules ’ structure was responsible for the differences in the molecular conformation changes. The molecules ’ interaction in the single-component protein affected secondary conformation of the proteins’ molecule. The proteins’ concentration ratio and the interaction were different in degree of molecular conformational change. These data demonstrates an example of combination of fluorescence spectrum experiment with Fourier transform infrared spectroscopy in the study of protein structura.