Abstract: 【Objective】 To screen and obtain the strains with antagonistic effects on the pathogens of alfalfa root rot，and optimize fermentation conditions，so as to provide strain resources for the biological control of alfalfa root rot.【Method】 The rhizosphere soil of alfalfa in Qinghai-Tibet Plateau was taken as the research object，and 5 pathogens of alfalfa root rot were used as the target to isolate bacteria by dilution coating method.The strains with antagonistic effects on alfalfa root rot pathogens were screened by plate confrontation method.The antibacterial activities of the antagonistic strains against 5 kinds of pathogens of alfalfa root rot were measured by plate confrontation method and mycelial growth rate method.Morphological observation，physiological and biochemical measurements and 16S rDNA sequence analysis were used to determine the taxonomic status of the antagonistic strains.The fermentation conditions of antagonistic strains were optimized by single factor test and orthogonal test.【Result】 92 strains of bacteria were isolated and purified from the rhizosphere soil of alfalfa，among which 16 strains with strong antagonistic effects against the tested pathogens were screened，among which strain LJ3-1 showed the strongest antifungal effect and broad spectrum antifungal activity.The antifungal activity of the filtrate of strain LJ3-1 showed that the inhibitory rate of the filtrate was 44.9%-77.4%.Strain LJ3-1 was identified as Bacillus subtilis based on colony morphological characteristics，physiological and biochemical measurements and 16S rDNA sequence analysis.The optimized fermentation conditions of strain LJ3-1 showed that the optimal fermentation medium was 3% glucose，1% yeast extract and 0.5% NaCl.The optimal fermentation conditions were as follows： initial pH 6.5-7.0，incubation temperature 35℃，250 mL conical bottle volume 80 mL，inoculum volume 3%，incubation time 36 h and rotation speed 180 r/min.Under the optimized fermentation conditions，the antifungal activity of strain LJ3-1 against Fusarium avenaceum increased from 76.5% to 89.3%.【Conclusion】 Strain LJ3-1 has good antagonistic effects against the 5 pathogens of alfalfa root rot，and it can be used as a candidate strain for the development of biological control agents of alfalfa root rot.