This study highlights the possibility of using brewers’ grains (BSGs) for the successive extraction of the main lignocellulosic biopolymers, namely, cellulose, hemicelluloses and lignin. An exhaustive chemical characterisation revealed a variability of composition in distinct batches of BSGs, depending on their origin and the brewing process used. In particular, the protein content can vary from 13wt% to 23wt%, which is accompanied by a change in the hemicelluloses content from 9% to 23% (in the samples of our study). By applying a two-step aqueous treatment, involving an acid (1.25% v/v aq. H2SO4) and a base (3% w/v aq. NaOH) at a temperature of 120°C and fixed reaction time of a few tens of minutes (15–90 minutes), more than 80% of hemicelluloses could be recovered. Cellulose could be isolated at more than 68%, while a high purity lignin could be recovered from a lignin-rich fraction (70wt%). Our work also suggests that the variability of the chemical composition of these BSGs is a hindrance to achieving process standardisation and large-scale exploitation. The pooling of various materials is therefore not a recommended option, and the preliminary chemical analysis of the composition is therefore a prerequisite for an efficient extraction process.