An Aspergillus flavus with the ability to yield high production of β-1,3-1,4-glucanase was found. The fermentation conditions for this enzyme in the shake flask were optimized using a combination of one-single-factor method and response surface methodology. The results indicated that the optimal culture medium components for β-1,3-1,4-glucanase production were 19g/L wheat bran, 30g/L (NH₄)₂HPO₄, 21g/L Tween-60,5g/L NaCl, 0.5g/L MgSO₄·7H₂O and 0.75g/L KH₂PO₄ at an initial pH of 8.0. The highest enzyme activity of 155.9U/mL was obtained when the strain was cultured at 38℃ for 6d in the optimized culture medium. The crude β-1,3-1,4-glucanase from Aspergillus flavus could hydrolyze oat and barley powder to mainly yield glucose. These results indicated that the extracellular β-1,3-1,4-glucanase produced by Aspergillus flavus possess great potential for its industrial applications.