The work aims to improve the effect of polysaccharide on protein food storage by exploring the affecting mechanism of polysaccharide on protein structure with chitosan and casein as research objects. Fluorescence spectroscopy and Fourier transform infrared spectroscopy were used to study the binding reaction between chitosan and casein under different pH conditions, and the binding constant, number of binding sites and binding force were calculated by mathematical equations. Under different pH conditions, chitosan quenched the fluorescence of casein, which belonged to static quenching. The quenching constant reached the maximum value of 4.702×103 L/mol at pH=5.2, and the binding constant reached the maximum value of 9.914×103 L/mol. Thermodynamic analysis showed that the spontaneous binding reaction was an exothermic reaction, and the main binding driving force was electrostatic interaction. Synchronous fluorescence spectroscopy and three-dimensional fluorescence spectroscopy indicated that the interaction of chitosan and casein could change the spatial conformation of casein. Fourier transform infrared spectroscopy illustrated that the addition of chitosan and the change of pH led to changes in the secondary structure of casein. The combination of chitosan and casein leads to structural changes in casein, which provides some theoretical basis for protein-polysaccharide interactions and also provides some experimental basis for the storage and development of dairy products based on protein-polysaccharide.