Enzymolysis kinetics, thermodynamics and molecular conformation of brewer's spent grain protein (BSGP) pretreated with ultrasound were examined. Results showed that ultrasound pretreatment significantly reduced Michaelis constant (K_m) and the thermodynamic parameters, including activation energy (E_a), enthalpy (ΔH), entropy (ΔS) and Gibbs free energy (ΔG) of BSGP during enzymolysis. Moreover, the initial reaction rate (V_i), association constant (K_a) and reaction rate constants (k) of BSGP pretreated with ultrasound were higher than those of native BSGP. The spectral analysis with Ultraviolet-visible (UV–vis) and Fourier transform infrared (FTIR) manifested that ultrasound pretreatment exposed hydrophobic groups and induced molecular unfolding of BSGP. Particle size analysis and scanning electron microscopy (SEM) showed that ultrasound pretreatment decreased particle size while increasing flexibility of BSGP. All these structural changes resulted in the improvement on enzymolysis efficiency of BSGP pretreated with ultrasound. Therefore, ultrasound pretreatment could be an efficient method to enhance the enzymolysis sensibility of BSGP.