Lignin‐based nanomaterials fabricated by solution self‐assembly in organic–aqueous solvent mixtures are among the most attractive biomass‐derived products. To accurately control the structure, size, and properties of lignin‐based nanomaterials, it is important to achieve fundamental understanding of its dissolution and aggregation mechanisms. In this work, atomic force microscopy (AFM) and molecular dynamics (MD) simulations are employed to explore the dissolution and aggregation behavior of enzymatic hydrolysis lignin (EHL) in different organic–aqueous solvent mixtures at molecular scale. EHL was found to dissolve well in appropriate organic–aqueous solvent mixtures, such as acetone–water mixture with a volume ratio of 7:3, whereas it aggregated in pure water, ethanol, acetone, and tetrahydrofuran. The interactions between the EHL‐coated AFM probe and the substrate were 1.21±0.18 and 0.75±0.35 mN m−1 in water and acetone, respectively. In comparison, the interaction decreased to 0.15±0.08 mN m−1 in acetone–water mixture (7:3 v/v). MD simulations further indicate that the hydrophobic skeleton and hydrophilic groups of lignin could be solvated by acetone and water molecules, respectively, which significantly promoted its dissolution. Conversely, only the hydrophobic skeleton or the hydrophilic groups were solvated in organic solvent or water, respectively, inducing serious aggregation of lignin.