The safety and in vitro probiotic properties of Lactobacillus reuteri isolated from a 128-year-old woman were analyzed in this study. Forty-nine strains of Lactobacillus reuteri were screened for their tolerance to simulated gastrointestinal fluid, and three strains with a survival rate of more than 90.00% were obtained: LT018, LT037, and LT046. The analyses of surface characteristics, antimicrobial activity, and safety of these three Lactobacillus reuteri strains were investigated, and the results showed that LT018 had relatively good adhesive reproductive capacity (hydrophobicity: 54.65% and auto-aggregation: 67.20%), and the surface hydrophobicity was negatively correlated with agglutination of the strains (R = -0.869). The non-acidic antibacterial substances could exert antibacterial action under acidic conditions, and the supernatant of these three strains had obvious inhibitory effects against Escherichia coli, Staphylococcus aureus, Bacillus cereus, Salmonella typhi, and Shigella flexneri. In particular, the strongest inhibitory effect against Staphylococcus aureus was observed, with an inhibition zone diameter of more than 15.00 mm. The strains did not produce harmful biogenic amines, and were sensitive to four types of commonly used antibiotics (erythromycin, tetracycline, gentamicin, and vancomycin), but were resistant to chloramphenicol. Based on the above results, these strains are believed to have a good potential for development.