Abstract: In order to gain a deeper understanding of the changes in moisture and protein structure during beef storage, the preservation effects of different natural preservatives (0.5% ε-polylysine, 0.5% tea polyphenols, 0.5% chitosan, and 0.2% ε-polylysine+0.2% tea polyphenols+0.2% chitosan) during beef storage period at 4 ℃ (12 d) were investigated in this study. Total volatile base nitrogen (TVB-N), water-holding properties, water distribution and migration, protein secondary structure, hydrophobicity, and other indexes were determined. Results showed that natural preservatives could significantly reduce TVB-N (P<0.05) and reduce storage losses of beef during storage, there was no significant advantage in pressure loss between 0.5% ε-polylysine, 0.5% tea polyphenols, and 0.5% chitosan treatment groups, while composite natural preservative significantly reduced pressure loss during mid-storage period (P<0.05). Natural preservatives were able to slow down the magnitude and rate of conversion of bound water to immobile water and the magnitude of conversion of immobile water to free water, as well as delay the process of bound water migration. Natural preservatives could maintain the stability of protein secondary structure (P<0.05), significantly reduce the exposed content of hydrophobic groups (P<0.05), and alleviate the protein degradation. There was no significant difference in the maintenance of myofibrillar protein among the 0.5% ε-polylysine, 0.5% tea polyphenols and 0.5% chitosan treatment groups, and composite natural preservative was significantly better than the individual natural preservative treatment groups in maintaining the structure of myofibrillar protein. In conclusion, composite natural preservative had the best preservation and water retention effects. The research could provide a scientific theoretical basis for the application and development of composite biological preservatives in terms of preservation and water retention.