In fermented food production, lactic acid bacteria (LAB) are the most commonly used organism, as they have been extensively used as probiotics since the last decade. In order to identify a new starter culture, we isolated and characterized LAB from alfalfa (Medicago sativa) forage, then evaluated their potential probiotic properties. Among ten isolates, one, namely KCC-34, showed significant cell growth and antifungal activity, and was thus selected for further characterization. The biochemical, physiological, and molecular (16S rRNA sequencing) properties of the selected isolate confirmed that the isolate is Lactobacillus plantarum KCC-34. The isolated strain showed a more significant susceptibility against various commonlyused antibiotics. L. plantarum KCC-34 exhibited greater antibacterial activity against Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, and Pseudomonas aeruginosa, and had strong antifungal activity against Aspergillus clavatus, Penicillium chrysogenum, and A. flavus. In addition, the L. plantarum KCC-34 strain also had the advantages of cell surface hydrophobicity and aggregation properties. Further, the L. plantarum KCC-34 strain proved its ability to survive in the stomach and in the small intestine. Besides, the new L. plantarum KCC-34 strain showed good DPPH free radical scavenging activity. Taken together, these features suggest that the novel L. plantarum KCC-34 can be potentially useful as a starter culture in fermented food production