BACKGROUND Beer is the most popular alcoholic beverage worldwide. In the manufacture of beer, various by-products and residues are generated, and the most abundant (85% of total by-products) are spent grains. Thanks to its high (hemi)cellulose content (about 50% w/w dry weight), this secondary raw material is attractive for the production of second-generation biofuels as butanol through fermentation processes. RESULTS This study reports the ability of two laccase preparations from Pleurotus ostreatus to delignify and detoxify milled brewer's spent grains (BSG). Up to 94% of phenols reduction was achieved. Moreover, thanks to the mild conditions of enzymatic pretreatment, the formation of other inhibitory compounds was avoided allowing to apply the sequential enzymatic pretreatment and hydrolysis process (no filtration and washing steps between the two phases). As expected, the high detoxification and delignification yields achieved by laccase pretreatment resulted in great saccharification. As a fact, no loss of carbohydrates was observed thanks to the novel sequential strategy, and thus the totality of polysaccharides was hydrolysed into fermentable sugars. The enzymatic hydrolysate was fermented to acetone-butanol-ethanol (ABE) by Clostridium acetobutilycum obtaining about 12.6 g/L ABE and 7.83 g/L butanol within 190 h. CONCLUSIONS The applied sequential pretreatment and hydrolysis process resulted to be very effective for the milled BSG, allowing reduction of inhibitory compounds and lignin content with a consequent efficient saccharification. C. acetobutilycum was able to ferment the BSG hydrolysate with ABE yields similar to those obtained by using synthetic media. The proposed strategy reduces the amount of wastewater and the cost of the overall process. Based on the reported results, the potential production of butanol from the fermentation of BSG hydrolysate can be envisaged.