The study aimed to investigate the biological properties of the protein fraction of brewers’ spent grain (BSG) and its sediments as well as the techno-functional properties of BSG protein (BSGP). BSG was incubated with 0.5% protamex and a combination of protamex and flavourzyme, in addition to the control (incubation without protease). The results showed that enzymatic treatment enhanced the antioxidant activity of BSGP. Compared to the sediment fraction, BSGPs had higher antioxidant capacities than those in sediments. The current study demonstrated that the FRAP value is aligned with the amount of the polyphenolic compounds, while BSGP is responsible for ORAC and ABTS capabilities. Enzyme treatment on BSG enhanced the antioxidant properties of BSGPs and the amount of the phenolic compounds of the sediments. BSGPs treated with proteases possessed higher oil-holding capacity, foaming properties and lower emulsion capability. In conclusion, enzymatic treatment of BSG enhanced the protein functionality and bioactivity as well as intensified the antioxidant activity of its sediments allowing further valorization.