Abstract: In this study, we systematically evaluated the in vitro characteristics of Bacillus velezensis SL to assess its potential as a probiotic. The probiotic properties of strain SL were systematically evaluated using a simulated gastrointestinal tolerance test, antibiotic sensitivity test, cell surface characteristic analysis, antioxidant activity determination, bacteriostatic ability test, and metabolomics technology. The results showed that the survival rate of strain SL in the simulated intestinal fluid was>80%. However, gastric acid tolerance was poor (120 min survival rate was 4.11%), and tolerance to high concentrations of bile salt (0.6%) was good. In addition, strain SL was sensitive to seven antibiotics and did not harbor any resistance genes. The hydrophobicity of the bacteria was 19.08%, and the self-aggregation rate at 12 h was 86%, indicating strong intestinal colonization potential. In the antioxidant experiment, the scavenging rates of strain SL on hydroxyl radicals (·OH) and superoxide anions (O₂⁻·) were 23.84% and 34.18%, respectively, and the scavenging rates of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⁺) radical were 37.40% and 36.52%, respectively, indicating moderate free radical scavenging abilities. Strain SL showed a certain bacteriostatic effect on pathogenic bacteria such as Salmonella, Clostridium perfringens, and Staphylococcus aureus, and exhibited a good inhibitory effect on Gram-positive bacteria. Metabolomic analysis identified both volatile (such as isobutyric acid and 2-methylbutyric acid) and nonvolatile (such as 9-nitrooleic acid, danye-emodin, and palmitoylglycine) metabolites with potential benefits for digestive enzyme activity and intestinal development, as well as anti-inflammatory, antimicrobial, and antioxidant properties. These results indicate that strain SL has the potential to be developed as a microecological agent. However, the stability of gastric acid should be enhanced by embedding or strain improvement to optimize its application performance.