β-glucanase can have a great influence on rheological properties and gluten protein structure of dough with a high oat content. We tested the farinographic properties of the pre-mixed oat bread flour, the dynamic oscillatory rheology of the dough, the content and average molecular weight of β-glucan, the free sulfhydryl content and proteins extractability in SDS solution of oat dough. We analyzed the secondary structure, SDS-PAGE protein patterns and surface hydrophobicity of the proteins in the dough. The results showed that, compared with the control group, after the addition of β-glucanase, the water absorption of the dough with 50% oat content, the storage modulus (G') and loss modulus (G') all decreased significantly. The gel-forming properties of the β-glucanase in oat itself was weakened. The deformability and fluidity of the dough were improved. As a result, the processing suitability of the dough is enhanced. The β-glucanase reduced the degree of polymerization of β-glucan in the dough. The average molecular weight decreased from 350 KDa to 62 kDa, the non-covalent interaction between gluten proteins was strengthened, the surface hydrophobicity of gluten proteins was reduced, and the protein. The secondary structure is converted from random coils and β-turns to β-sheets, resulting in increased hydration and aggregation of gluten. The above research showed that β-glucanase had great potential on improving the processing adaptability of high oat content dough.