The 26 strains isolated from traditional fermented yak milk in Yangbajin-Tibet and Yunnan-Shangrila area. In this study, the inhibitory spectrum of the selected strain was determined by Oxford cup double agar diffusion method Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Yeast was taken as the indicator, to screen out lactic acid bacteria with high antibacterial activity from various stains. At the same time, according to the exclusion of organic acids, hydrogen peroxide and proteolysis detection, speculated that the inhibitory effect of lactic acid bacteria may come from lactobacillin. The results showed that 3, 21, 23, 24, 25 strains have higher antibacterial activity. And 5 strains were identified by 16S rDNA sequence analysis. The 3, 23 and 24 as Lactobacillus casei, 21 as Lactobacillus plantarum, 25 as Lactococcus lactis subsp. lactis. Antibacterial spectrum test showed that lactobacillin could effectively inhibit some food-borne gram positive and negative bacteria. All isolated strains had no antimicrobial activity against fungi.