Abstract To reveal the synergistic effect of hydrogen peroxide and ammonia on lignin, four kinds of ball-milled wood lignins, named as UN-L, HP-L, AFEX-L and H-AFEX-L respectively, were isolated from corn stover of untreated, hydrogen peroxide pretreatment alone, ammonia fiber expansion pretreatment alone and combined hydrogen peroxide and ammonia fiber expansion pretreatment. Modern analytical techniques including elemental analysis, gel permeation chromatography (GPC), Fourier transform infrared (FT-IR) spectroscopy and two-dimensional heteronuclear single-quantum coherence nuclear magnetic resonance (2D-HSQC NMR) spectroscopy were applied to analyze the characteristics of the obtained lignins. The results showed that oxidation, ammonolysis were main chemical reaction during H-AFEX pretreatment. The action site of oxidation was S unit, while that of ammonolysis were ferulate and p-coumarate ester bonds. The synergistic effect of hydrogen peroxide and ammonia strengthened ammonolysis effect whereas weakened oxidation effect. Ammonia selectively degraded the ester bond, while hydrogen peroxide selectively degraded the resinol structure. The synergistic effect of hydrogen peroxide and ammonia could not only effectively degrade lignin, but also greatly reduce its PI to obtain homogeneous lignin fragments.