The rising protein demand has driven intensified research into alternative protein sources and extraction technologies. Brewer’s spent grain (BSG), which is rich in protein, remains mostly underutilized. This study aimed to optimize BSG protein extraction conditions using pulsed electric field (PEF) by assessing the influence of pulse numbers (5000–9000), electric field strength (8–10 kV/cm), and frequency (8–10 Hz) on protein recovery and purity. The optimized conditions (5386 pulses, 10 kV/cm field strength, and 10 Hz frequency) provided a higher extraction yield with a significant improvement of approximately 90% (p < 0.05). Essential amino acids in proteins extracted via PEF were significantly increased (60,864.84 mg/100 g), particularly phenylalanine, threonine, and valine; furthermore, amino acid score (AAS) and protein digestibility-corrected amino acid score (PDCAAS) were found to be superior to those of protein obtained through alkaline extraction. The PEF treatment resulted in the modification of the secondary structures of proteins from α-helices and β-turns to β-sheets, as well as the enhancement of the hydrophobic−hydrophilic amino acid balance. The functional properties of the proteins, particularly their foaming properties and solubility, were significantly affected by PEF (p < 0.05). In conclusion, PEF-assisted extraction produces high-quality BSG protein concentrates efficiently while rendering the process environmentally sustainable.