In recent years, more and more functional activities have been discovered with the in-depth study of the monascus pigment, but some toxic effects of the monascus pigment have raised questions about its safety. Therefore, it is important to elucidate the interaction between monascus pigments and macromolecules in human body for further study of their transport, metabolism and toxic side effects. Spectroscopy is an effective method to study the interaction of small molecules with proteins in solution. It has been widely used in research for its high sensitivity, strong selectivity, low sample size, and simple method. In this study, rubropunctamine(Rub) was taken as the typical representative of monascus pigment to research the interaction of Monascus pigments with macromolecules bovine serum albumin(BSA). The fluorescence quenching effect of different concentrations of Rub on BSA was investigated by endogenous fluorescence spectroscopy and synchronous fluorescence spectroscopy. Then Stern-Volmer equation, the Lineweaver-Burk function and the Van’t-Hoff equation were used to determine the type of action, the number of binding sites and the interaction mechanism of BSA and Rub. The effect of Rub on the BSA secondary structure was quantitatively determined by circular dichroism spectrum. Finally, using the computer to perform the molecular docking simul- ation on the interaction of Rub and BSA. The results show: (1) Rub has a strong fluorescence quenching effect on BSA, and endogenous fluorescence spectrum shows that endogenous fluorescence decreases by 306.1 and emission wavelength shifts by 6.8 nm. Synchronous fluorescence shows that fluorescence quenching mainly occurs on tryptophan residues. (2) The dynamic quenching rate constant Kq calculated by the Stern-Volmer equation is 2.335×1012 L·(mol·S)-1, which is much larger than the maximum diffusion collision constant allowed: 2.0×1010 L·(mol·S)-1, and the annihilation is a pure static quenching process. (3)The binding constants reach above 103 L·mol-1 which is calculated by the equation lg［(F0-F)/F］=lgK0+nlgcQ, and the number of binding sites is approximately 1. The apparent binding constant becomes smaller with increasing temperature. (4) Under different temperature, ΔH, ΔS and ΔG are less than zero, so the interaction can occur spontaneously and hydrogen bonding and van der Waals force are the main interaction forces. (5) the α-helical content in the secondary structure of BSA combined with Rub decreased from 29.4% to 20.2%; The β-fold increased from 39.9% to 50.7%; β-rotation decreased from 6.5% to 3.5%; The random coil increased from 24.2% to 25.6%. (6) Rub is located in the pocket formed by Arg458, Asp108, Glu424, Ser428 and other amino acids in BSA, and it has Van Der Waals force with Arg458 and hydrogen bond interaction with Arg144 which affects Trp213 microenvironment.