This study aimed to evaluate the adhesion of the elongation factor G (EF-G) from Lactobacillus salivarius to Staphylococcus aureus through gene recombination and gene knockout. The effect of prokaryotically expressed protein on the adhesion of S. aureus was examined. The results showed that EF-G increased the in vitro adhesion rate of S. aureus by 43% compared to the control group. As demonstrated by the ef-g gene knockout test using a mucin adhesion model method and fluorescent labeling, knockout of the ef-g gene did not affect colonization of the intestinal tract by L. salivarius Δef-g, and L. salivarius Δef-g reduced the in vitro and in vivo adhesion rate of S. aureus by 40% and 20%, respectively. These findings were validated by scanning electron microscopy (SEM). Therefore, we concluded that EF-G plays a role in promoting the adhesion of S. aureus, which provides a scientific basis for the application of probiotics.