Abstract: The combination of carrageenan and casein had a certain protective effect on the intestinal barrier. In order to study whether the digestive enzymes of the κ-carrageenan and casein in vitro digestion simulation were the main factors affecting the conformational transformation of polysaccharides or polysaccharide-protein composite systems, κ-carrageenan was used as the research object and the casein in cow's milk was selected as the target food matrix in this paper to explore the effect of digestive enzymes on the binding stability of κ-carrageenan/casein composite systems in vitro simulated digestion. The results showed that gastrointestinal digestive enzymes had little effect on the binding of κ-carrageenan to casein, but could significantly increase the exposure of sulfate groups in the system, which was not conducive to the configuration of κ-carrageenan. The casein in the system was decomposed into low molecular weight proteins or peptides, which increased the characteristic length of the composite system, weakened the interaction, decreased the viscoelasticity, and decreased the stability of the double helix structure. This study provides a theoretical basis for the safe application of κ-carrageenan.