A novel L. harbinensis VF was isolated from fermented vinegar and identified through biochemical characteristics and 16S rRNA sequencing analysis. Characteristics of probiotics were studied for acid and bile salt tolerances, hemolytic activity, antibiotic resistance, antimicrobial activity, cell surface hydrophobicity, and aggregation. The survival rates of the isolate were maintained at 68.9% and 95.6% after 3 h incubation at pH 2.0 and 2.5 and were over 74% at bile salt concentrations of 0.3% and 0.5%. The hemolytic activity was confirmed to be -hemolysis. The isolate showed broad antibiotics-resistance in over 12 antibiotics except for trimethoprim/sulfamethoxazole, compared to L. plantarum (KCTC 3108), used as control, and previously reported L. harbinensis. Antimicrobial activity was confirmed against pathogens, B. cereus, E. coli O157:H7, L. monocytogenes, and S. typhimurium, except S. aureus, and it was attributed to produced organic acids (low pH). The hydrophobicity for xylene and chloroform were 16.7% and 20.4%, respectively, lower than those of L. plantarum (41.2% & 49.9%). The auto-aggregation and coaggregation with pathogens were 83.8% and 49.2-87.6% at 24 h incubation, respectively, higher than those of L. plantarum. From this study, L. harbinensis VF is likely to be used as a probiotic strain and is a strong candidate for commercial application.