Lignin condensation reactions are hard to avoid or control during separation, which is a deterrent to lignin isolation and post‐conversation, especially for the full utilization of lignocelluloses. Selective protection of β‐aryl ether linkages in the isolation process is crucial to lignin valorization. Herein, a two‐step acid/alkali separation method assisted with l‐cysteine for eucalyptus lignin separation is developed, and the isolated l‐cysteine lignins (LCLs) are comprehensively characterized by 2D NMR, 31P NMR, thioacidolysis, etc. Compared to the two‐step control treatment, a much higher β‐O‐4 content is preserved without reducing the separation efficiency assisted by l‐cysteine, which is also significantly higher than alkali lignin and kraft lignin. The results of hydrogenolysis show that LCLs generate a much higher monomer yield than that of control sample. Structural analysis of LCLs suggests that lignin condensation reaction, to some extent, is suppressed by adding l‐cysteine during the two‐step acid/alkali separation. Further, mechanistic studies using dimeric model compound reveals that l‐cysteine may be the α‐carbon protective agent in the two‐step separation. The role of l‐cysteine in the two‐step lignin isolation method provides novel insights to the selective fractionation of lignin from biomass, especially for the full valorization of lignocellulosic biomass.