[Objective] To isolate and screen antagonistic strains with strongly inhibitory activity on Rhizoctonia solani (Linum usitatissimum L.) from the rhizosphere soil of healthy flax. [Methods] Antagonistic strains were screened out through plate coating and dual culture method. Strains were identified by morphological, physiological and biochemical characteristics and 16S rRNA gene sequence analysis and their biocontrol effect were determined by greenhouse experiment. The fermentation condition was optimized by single factor and Uniform design experiment. [Results] Strain HXP-5 showed strongly inhibitory activity on R. solani (Linum usitatissimum L.) and also antagonistic?effect against other seven phytopathogens. Strain HXP-5 was identified as Bacillus subtilis. Greenhouse experiment showed that the control efficiency were 71.22%. The optimized culture conditions produced antimicrobial active substances of strain HXP-5 were liquid volume 100 mL in 250 mL flask, 210 r/min at 27 °C, inoculation size of 1.7% for 72 h with a medium of 2.3% glucose, 0.25% tryptone+yeast extract (3:1), 0.18% NaCl. [Conclusion] The antagonistic strain HXP-5 to R. solani (Linum usitatissimum L.) was identified as B. subtilis. And it has a stronger effect on biocontrol of Flax Rhizoctonia solani. HXP-5 strain shows stronger antagonistic action to R. solani (Linum usitatissimum L.) under optimized fermentation conditions.