The hydrogen peroxide presoaking prior to ammonia fiber expansion (H-AFEX) pretreatment is an effective and promising method for agricultural residues to decrease biomass recalcitrance and enhance enzyme accessibility. To illuminate the structural changes of lignin after the H-AFEX process, ball-milled wood lignins were isolated from raw material and H-AFEXtreated corn stover. The features and structures of the obtained lignins were characterized by elemental analysis, gel permeation chromatography, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy. The results demonstrated that the H-AFEX-treated lignins had higher oxygen and nitrogen contents while lower carbon and hydrogen contents when compared to those of untreated lignin. A remarkable decrease in molecular weight of H-AFEX-treated lignin was observed. Ammonolysis, hydrolysis, and oxidation reactions were major chemical modifications to lignin, and the cleavages of ferulate and p-coumarate ester bonds, alkyl ether bonds, and aryl ether bonds were observed during the H-AFEX process. G unit lignin was more easily degraded through demethoxylation, while the interunit linkages of resinol and phenylcoumaran were relatively stable. The study on lignin structural changes during the H-AFEX process could reveal the pretreatment mechanism and develop of a new pretreatment method, with a perspective of reducing biomass recalcitrance and improving the bioconversion of biomass to biofuels or biomaterials.