The growth condition and analysis of acid production capacity showed that Pediococcus pentosaceus F28-8 had a good acid adaptability, and the surface structure was observed with transmission electron microscopy. The S-layer was extracted with 5mol/L LiCl and analysed by SDS-PAGE and MALDI-TOF-MS. It was found that the S-layer of F28-8 contained a kind of surface protein with molecular weight of 83.5kDa and 51.1kDa. The further surface property test, collagen adhesion test and antibacterial experiment showed that the stripping of surface protein significantly reduced the surface properties (automatic aggregation ability, surface hydrophobicity and surface charge), with a decrease of 19.0%-56.1%. The surface protein of F28-8 extracted by LiCl significantly reduced the adhesion of Salmonella and Staphylococcus aureus to collagen, with a decrease of 74.59% and 81.16%. The inhibitory rate of surface protein of F28-8 extracted by LiCl against Salmonella and Staphylococcus aureus reached 31.5% and 15.6%, respectively. The results indicate that surface protein of P. pentosaceus F28-8 can change the surface properties of cells, enhance their ability to inhibit the adhesion of pathogenic bacteria and exhibit certain antibacterial ability, and has certain application potential in the field of antibacterial agents.