Abstract: This study aimed to investigate Lactobacillus salivarius S54, a strain isolated from buffalo milk with potential probiotic properties, through whole-genome sequencing and phenotypic validation to systematically evaluate its safety, tolerance, and probiotic characteristics, thereby providing a theoretical basis for developing novel probiotic products. Methods: The complete genome of the strain was sequenced using third-generation sequencing technology. Antibiotic resistance genes and virulence factor genes were screened using the CARD and VFDB databases to assess its safety. Secondary metabolite biosynthesis gene clusters were predicted via genome mining tools. Functional annotation and tolerance-related gene analysis were performed using databases such as KEGG. In vitro experiments, including gastrointestinal fluid tolerance, antibacterial activity, antioxidant capacity, and prebiotic utilization assays, were conducted to validate its probiotic properties. Results: The genome of Lactobacillus salivarius S54 consisted of a circular chromosome of 1980939 bp with a GC content of 33.04%, and a predicted plasmid of 168151 bp. No known pathogenic virulence factors or antibiotic resistance genes were detected. A gene cluster encoding antimicrobial and antioxidative secondary metabolites was identified. Functional annotation revealed multiple genes associated with tolerance, and in vitro experiments confirmed its strong antibacterial and antioxidant activities. The strain exhibited high tolerance to high temperatures and gastrointestinal fluids, as well as the ability to utilize common prebiotics for growth. Conclusion: Lactobacillus salivarius S54 demonstrates favorable probiotic properties and a high safety profile, making it a promising candidate for the development of probiotic formulations or fermented dairy products, with significant potential for future applications.