Objective: The surface layer protein (SLP) of Lactobacillus， possesses adhesive functionalities， including facilitating the adherence of bacterial cells to intestinal epithelial cells or stable binding to surface constituents of cells. This study aimed to explore the adhesion properties of the SLP of Lactobacillus in Tibet kefir and evaluate its impact on the aggregation among microorganisms and biofilm formation. Methods: SLP-producing strains were selected from Tibet kefir using conventional separation techniques and an acidic lithium chloride extraction method. The impact of SLP on the hydrophobicity and surface charge of bacterial cells was investigated by employing the microbial adhesion to solvents (MATS) assay. Finally， the self-aggregation rate of Lactobacillus， their co-aggregation rate with yeasts， and the capacity for biofilm formation were determined to assess the influence of SLP on microbial aggregation and biofilm formation within the Tibet kefir microbiota. Results: 19 strains of SLP-producing Lactobacillus from Tibet kefir， with SLPs exhibiting an average molecular weight of approximately 70 ku， and the highest production level reaching 1.16 g/mL. By comparing the surface characteristics and aggregation abilities of bacteria before and after the removal of SLP， it was found that the removal of SLP caused the average auto-aggregation rate and co-aggregation rate of bacteria to decrease by 1.63%-7.56% and 0.96%-10.63%， respectively， and the reduction range of the average absorbance value corresponding to the growth of the mixed biofilm was 0.52-1.13 nm. Conclusion: Tibet kefir was found to harbor an abundant population of high SLP-producing strains， with the majority representing dominant microbial species. As a critical component governing aggregation and adhesion abilities， SLP plays a pivotal role in the biofilm formation and stable propagation of the microbial community within Tibet kefir.