ABSTRACT: An extreme thermostable and acidic tolerable β-glucanase was isolated and characterized from aerobic fungi Trichoderma koningii ZJU-T. The optimal reaction temperature and pH for the β-glucanase were 100 °C and pH 2.0, respectively. The β-glucanase showed increased stability at higher temperatures and lower pH values when compared to other β-glucanases. The optimum conditions for the β-glucanase stability were found to be pH 4.0 and 80 °C. Even subjected to 100 °C for 3 h, β-glucanase activity did not show significant reduction. Moreover, K+ significantly enhanced β-glucanase activity at the concentration of 1 mM, while EDTA and other metal ions such as Mg2+, Mn2+, Zn2+, Ca2+, Fe2+, Pb2+, and Fe3+ inhibited β-glucanase activity. Denaturants, including sodium dodecyl sulfate (SDS) and mercaptoethanol, also inhibited β-glucanase activity at a concentration of 5%. However, in the presence of 7 M urea, residual activity of the β-glucanase still remained 14.5%.