Seventeen endophtye strains were isolated from soybean seeds by enrichment culture, and three endophyte strains producing β-mannanase were screened using Congo red dye method. By the shaking-flask culture, a strain of bacterium with the highest enzyme activity of 54.59 U/mL was obtained, and it was identified as Bacillus subtilis by the analysis of morphological, physiological and biochemical characteristics and 16S rDNA sequences. Enzymic properties of the β-mannanase revealed that the optimal temprature and pH were 30 °C?50 °C and 7.0, respectively. The enzyme activity still remained 68% when the enzyme was treated at 50 °C for 2 h; and more than 64% enzyme activity was remained after 1 h treatment at pH 5.0?9.0. In addition, the enzyme can be activated by Zn2+, Co2+, Ba2+, K+, and Ca2+ which has the most significant effect on the enzyme activity to improve 31% of activity. But, Mn2+ and EDTA could inhibite the enzyme activity.