Probiotics were isolated and screened from yak dung and milk in Tibet by calcium transparent circle method, and identified through morphological observation and molecular biology techniques. Their growth characteristics, acid production capacity, bile salt, acid, artificial gastric juice resistance and inhibit ability to Escherichia coli were analyzed. The results showed that a total of 25 strains of lactic acid bacteria were isolated and screened, among them, 7 strains were identified as probiotics, which were 1 strain of Lactobacillus plantarum, 2 strain of Lactobacillus rhamnosus and 4 strains of Lactobacillus paracaseium. Among them, L. plantarum B2 had good growth advantages, acid production capacity (the final pH value of fermentation was about 4), acid resistance (pH 4), bile salt resistance (0.6%), artificial gastric juice resistance (survival rate 57.4%), and inhibitory effect on E. coli (inhibition zone diameter 22.0 mm).