The lignocellulosic materials produced after each step of a biorefinery plant using simultaneous saccharification and fermentation (SSF) technology on wheat straw (Triticum spp.) for bioethanol production were characterized by spectroscopic and chromatographic techniques in order to investigate the macromolecular interactions between the lignin and polysaccharides. In order to valorize the lignin cakes, a purification step was set up and the extraction conditions (acid pretreatment, temperature, time, and NaOH concentration) were optimized by a chemiometric approach in terms of yield and purity. Residual carbohydrate impurities, free and/or chemically bonded to lignin (lignin carbohydrates complexes), were individuate as the most critical factor for a satisfactory lignin extraction. Finally, the lignin samples collected according to the optimized extraction conditions were chemically characterized and low molecular weight, high phenols concentration, and low carboxylic acids content were recognized as interesting features for industrial applications.