Abstract: In order to study the interaction between betanin and whey protein isolate (WPI) and the effect of whey protein isolate on the thermal stability of betanin, WPI-Betanin complex was constructed by self-assembly method. UV-vis spectroscopy was used to verify the formation of complex. The mechanism of complex formation and the changes of protein structure were investigated by fluorescence spectroscopy and circular dichroism, and the complex was visualized by molecular simulation technology. The results showed that betanin and whey protein isolate formed a complex mainly through van der Waals force and hydrogen bond, and the number of binding sites was about 1. The formation of the complex resulted in increased turbidity and decreased surface hydrophobicity of the protein, and the quenching mechanism was static, with a quenching constant of 1.78×10³ L/mol at room temperature. The interaction changed secondary structure contents and the microenvironment of tryptophan and tyrosine in whey protein isolate. Moreover, the thermal stability of betanin was improved by WPI. The retention rate of betanin increased from 6.17% to 27.26% after heating at 80 ℃ for 1 h. This study provides a theoretical basis for the application and development of functional protein pigment complex and the color protection of betanin.