New β-1,3;1,4-glucanase was purified from Aspergillus niger US368. The pure glucanase has a molecular mass of about 32kDa. The N-terminal sequence of the purified enzyme (A-G-T-N-P-P-I-G-V) was determined. The optimum pH and temperature recorded for enzyme activity were 5 and 60°C, respectively. It also displayed marked thermostability with a half-life of 30min at 70°C. At 37°C, the enzyme showed 100% stability from pH 3 to 10. The Km and Vmax values exhibited by the enzyme on barley β-glucan were 0.62mgml−1 and 34.46Uml−1, respectively. The enzyme is a retaining-one and was only active toward glucan containing β-1,3;1,4-linkages. The production of β-glucanase with barley flour as the sole carbon source was optimized. This is the first report on the purification and characterization of a β-1,3;1,4-glucanase from A. niger. This lichenase could be considered as a candidate for future application particularly in the animal feed industry.