The optimization of cultural conditions for β-glucanase production by Bacillus subtilis ZJF-1A5 was investigated in flask trials. Temperature had gre at effect on β-glucanase production which maximized at optimal temperature of 37 ℃ and decreased significantly when temperature was over 37℃.Charge quantity af fected β-glucanase production significantly. Adding oxygen vector N-dodec ane or acetic ether benefited β-glucanase production, but it depended on the conc entrat ion and charge quantity. The results of fractional factorial design showed that age and size of inoculum and shaking speed were the key factors affecting β -gluc anase production and the cultivation time span to reach the highest β-gluc anase activity. The optimal cultural conditions for β-glucanase production obtai ned wi th CCD were as follows: inoculum age and size (16 h, 3.82%(v/v)), shaking speed 2 10 r/min, charge quantity of 30 mL in 250 mL flask and initial pH 7.0, cultured at 37℃ for 50 h. Repeated experimental results accorded with those predicted b y a second-order polynomial model. The amount of β-glucanase, α-amylase and neut ral protease produced by B subtilis ZJF-1A5 was associated partially with c ell g rowth. Those three enzymes' activities increased following the cell growth and i ncreased significantly when cells entered the stationary phase.