The study focused on the structural sensitivity of lignin during the phosphoric acid-acetone pretreatment process and the resulting hydrolysis and phosphorylation reaction mechanisms using density functional theory calculations. The chemical stabilities of the seven most common linkages (β-O-4, β-β, 4-O-5, β-1, 5-5, α-O-4, and β-5) of lignin in H3PO4, CH3COCH3, and H2O solutions were detected, which shows that α-O-4 linkage and β-O-4 linkage tend to break during the phosphoric acid-acetone pretreatment process. Then α-O-4 phosphorylation and β-O-4 phosphorylation follow a two-step reaction mechanism in the acid treatment step, respectively. However, since phosphorylation of α-O-4 is more energetically accessible than phosphorylation of β-O-4 in phosphoric acid, the phosphorylation of α-O-4 could be controllably realized under certain operational conditions, which could tune the electron and hole transfer on the right side of β-O-4 in the H2PO4- functionalized lignin. The results provide a fundamental understanding for process-controlled modification of lignin and the potential novel applications in lignin-based imprinted polymers, sensors, and molecular devices.