Production, purification, and characterization of a novel β-1,3-1,4-glucanase (lichenase) from thermophilic Rhizomucor miehei CAU432 were investigated. High-level extracellular β-1,3-1,4-glucanase production of 6230 U/mL was obtained when oat flour (3%, w/v) was used as a carbon source at 50 °C. The crude enzyme was purified to homogeneity with a specific activity of 28818 U/mg. The molecular weight of purified enzyme was estimated to be 35.4 kDa and 33.7 kDa by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature of the enzyme were pH 5.5 and 60 °C, respectively. The K(m) values of purified β-1,3-1,4-glucanase for barley β-glucan and lichenan were 2.0 mM and 1.4 mM, respectively. Furthermore, the gene (RmLic16A) encoding the β-1,3-1,4-glucanase was cloned and its deduced amino acid sequence showed the highest identity (50%) to characterized β-1,3-1,4-glucanase from Paecilomyces thermophila. The high-level production and biochemical properties of the enzyme enable its potential industrial applications.