Abstract: The high propionate-producing strains were isolated and screened from fresh raw milk, and experiments such as morphological characteristics, physiological and biochemical and sugar fermentation tests, 16S rDNA gene sequence homology analysis, and multilocus sequence typing were performed to identify the strain. The utilization rate and acid production of different carbon sources were analyzed, and the safety of the strain was evaluated from the aspects of hemolysis test and antibiotic resistance test. Results showed that:A total of 54 strains capable of producing propionic acid were isolated from 4 batches of samples, and the propionic acid yield of one strain reached 7.38 g/L, which was Propionibacterium freudenreichii. The effects of glucose and glycerol on their growth were compared, and it was found that the utilization of glucose was greater than the utilization of glycerol. The yield of propionic acid reached 7.38 g/L after anaerobic culture at 30℃ for 120 h in glucose-containing medium. The strain is non-hemolytic, resistant to aminoglycoside antibiotics such as kanamycin, sensitive to ampicillin, vancomycin, chloramphenicol, clindamycin, tetracycline, and mediated by erythromycin. In summary, Propionibacterium freudenreichii B1 has certain potential application value.