Abstract: To improve the quality of fermented beef jerky and develop healthy fermented meat products with a good palatability, Lactobacillus plantarum ST3.5, Lactobacillus pentosus S4.3, and Weissella cibaria L4.2 were used in this study as enhanced starter cultures and added to beef as a single strain. By measuring the indices of protein advanced structure, myofibril microfibril index (MFI), and microstructure, this study investigated the effects of three lactic acid bacteria on the physicochemical property and structural characteristics of fermented beef jerky. The results showed that all three Lactobacillus strains could improve the quality of fermented beef jerky compared to natural fermentation. In particular, Lactobacillus pentosus S4.3 increased the TCA-soluble peptide content (15.66 mg/g) and MFI value (107.45), and decreased the shear stress (40.45 N) of the fermented beef jerky products. At the same time, the relative content of α-helix decreased, and the relative content of β-sheet and random coil increased. Scanning electron microscopy showed that fermentation of Lactobacillus pentosus S4.3 could effectively destroy the integrity of muscle fibers, and the degree of fiber fragmentation was the greatest. In conclusion, Lactobacillus pentosus S4.3 could improve the quality of fermented beef jerky products by effectively promoting the unfolding of protein structures and fragmenting beef muscle fibers.