Abstract: In this paper, SDS-PAGE had determined the best dissociating agent for extracting surface layer proteins from Lactobacillus acidophilus JCM 1132 in the 5 mol/L LiCl, 4 mol/L guanidine hydrochloride, 8 mol/L urea, 5 mol/L LiCl combined with 1 mol/L LiCl. The effect of different treatment methods on the surface characteristics of Lactobacillus was further studied. The results shoeed that two different concentrations of LiCl was the chosen as the best dissociating agent for extracting surface layer proteins, and this method could extract high purity surface layer proteins. And after using this method to remove the surface layer proteins, the survival rate of the L. acidophilus was the highest, about 99.27%. The surface charge, surface hydrophobicity, self-aggregation and co-aggregation with Salmonella were all reduced after the removal of the surface layer proteins by different methods. It showed that surface layer proteins played a key role in the surface properties of L. acidophilus and positively influence the probiotic function of the bacterium.