The conformational properties of hydrolysates derived from brewer’s spent grain protein (BSGP) pretreatment with and without ultrasound were investigated, and the effect of BSGP hydrolysates (BSGPH) supplementation on the brewer’s yeast growth and fermentation performances were examined. Results demonstrated that both BSGPH had substantial influences on the biomass, viability, sugar consumption and ethanol production of yeast. Specifically, medium supplemented with hydrolysates derived from BSGP pretreated with ultrasound (UBPH) for 4 h hydrolysis (UBPH-4) increased biomass by 7.7%, viability by 9.78%, sugar consumption by 37.16%, and ethanol yields by 20.20% (v/v), respectively, compared with the blank. UBPH-4 exhibited a looser morphology and disordered secondary structure, with a decrease in α-helix and increased random coil. Meanwhile, a higher proportion of short peptides and hydrophilic amino acids in UBPH-4 might account for its preferred bioactivity. All findings revealed that UBPH were effective nitrogen sources for yeast growth and fermentation.