As a newly-developed method for pretreating lignocellulosic biomass, phosphoric acid plus hydrogen peroxide (PHP) was employed as a pretreatment solvent to fractionate wheat straw. The structural properties of the derived lignin were elucidated in particular. Results indicated that 100.0 g of wheat straw (dry basis) yielded 39.7 g of cellulose-rich fraction, 4.7 g of oligosaccharides, and 4.5 g of lignin at mild conditions of 50 °C within 1.0 h. The resultant cellulose-rich fraction was highly accessible to hydrolytic enzymes with 88-96% cellulose-glucose conversion in 24 h, suggesting a great potential for producing biofuels. The derived lignin was characterized as having high purity (≤1.0% residual carbohydrates) and relatively low molecular weight (Mw < 1436 g mol-1) and being abundant in carboxylic acid functional groups. According to the 31P, 13C, and 2D heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy results on the derived lignin, the degree of condensation was quite limited during PHP pretreatment, the oxidants, produced as HO+ or HO· in pretreatment, were substantially responsible for the extensive ring-opening on the aromatic substructure. The obtained results offered the technical feasibility of fractionating lignocellulosic biomass using PHP and a better understanding of the delignification mechanisms for PHP pretreatment.