Abstract: The congo red staining method was used to screen β-mannanase producing strains with high yield. A strain MM5 was isolated from soil. The stain MM5 was identified as Bacillus subtilis according to the results of physiological and biochemical tests as well as 16S rDNA sequence analysis. The activity of β-mannanase by strain MM5 achieved 1 594 U/mL. After the strain MM5 was treated with ultraviolet radiation, a mutant strain LD24H4 was obtained and its activity increased to 3 717 U/mL. Our experiment adopted the single-factor design and orthogonal design. According to their results, the optimal culture medium (per liter) of LD24H4 for β-mannanase production was determined as 20.0 g konjac powder, 2.5 g casein, 1.0 g NaCl, 0.5 g MgSO4, 0.5 g KH2PO4, pH 7.5. The optimal conditions for fermentation were obtained as the temperature of 47 ℃, inoculation volume of 1%, 90 mL of culture medium in a 250 mL flask, and rotation speed of 200 r/min. Under the optimal conditions for 26 hrs, the β-mannanase activity increased to 12 534 U/mL, 3.37 times as high as the original activity.