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Brewer’s spent grain (BSG) is the major byproduct of the brewing industry. Although BSG contains 20–30% protein, the high fiber content (50–70%) makes it difficult to be used as a food ingredient. Therefore, BSG is used as a low-value ruminant feed or buried in landfills, causing substantial resource losses. In this study, an integrated process was developed to produce protein hydrolysates and 2,3-butanediol (2,3-BDO) from BSG, aiming to holistically utilize BSG with minimal secondary waste generation. BSG was first subjected to enzyme-assisted fractionation to separate protein from fiber residues. The fiber residue was then pretreated and hydrolyzed to obtain sugars. Finally, sugars were fermented to 2,3-BDO by the newly isolated thermophilic and alkaliphilic Bacillus licheniformis YNP5-TSU. The integrated process could be used to recover over 80% of protein from BSG to produce protein hydrolysates with a …