The high β-glucan content in barley disrupts the gluten network in dough. Scanning electron microscopy (SEM), differential scanning calorimetry (DSC), fourier transform infrared spectroscopy (FTIR), and solid-state nuclear magnetic resonance (NMR) techniques were used to clarify how β-glucan affected the quality of the gluten network structure with β-glucan contents of 0–2%. The results suggest that the physical hindrance of the β-glucan gel destroyed the formation of the gluten network structure. When 1.0–2.0% β-glucan was added, the percentage of α-helical structures increased significantly. When the added amount of β-glucan reached 2.0%, the sulfhydryl group (SH) content increased from 8.06 to 10.27 µmol/g, and the disulfide bond (SS) content decreased from 240.09 to 217.38 µmol/g. The interaction between β-glucan and gluten mainly resulted from the interaction of electron-withdrawing groups, such as carbonyl groups (CO) and double bond carbons (CC), and carbon atoms on the side chains of β-glucan, which play an important role in the central structure of glutenin.