Abstract: The objective of this study was to investigate the effect of rice wine on the quality of beef jerky. The beef jerky fermentation process was optimized by determining the moisture content, color difference, pH, shear force, texture, and water activity, with sensory evaluation scores serving as the response variables in a response surface methodology experiment. Additionally, changes in myofibrillar proteins during fermentation were thoroughly examined using solubility measurement, surface hydrophobicity assessment, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal processing conditions were rice wine addition, 6%; fermentation time, 24 h; and baking time, 2.5 h. The beef jerky produced under these conditions exhibited physicochemical properties with moisture content, 42.39%; pH, 5.61; water activity, 0.9298; and shear force, 28.97 N. The fermented beef jerky prepared using this process had excellent color, moderate texture, unique flavor, and high overall quality. During the fermentation process, the solubility of myofibrillar proteins in beef gradually decreased with increasing fermentation time, whereas its surface hydrophobicity continuously increased. This phenomenon indicated that the fermentation promoted the gradual unfolding of the highly aggregated structure of myofibrillar proteins, resulting in the exposure of internal hydrophobic groups. The SDS-PAGE results demonstrated that the addition of rice wine was more conducive to the degradation of myofibrillar proteins. The findings of this study provide important theoretical support for the development of new fermented beef jerky products, and offer new insights for research in related fields.