Lignin nanoparticles (LNPs) are gaining increasing interest for applications in various fields, where the particle homogeneity, morphology, and surface properties are critical for performance. In this study, lignin obtained via kraft process from spruce and eucalyptus was employed as precursor for the fabrication of lignin nanoparticles with tunable physicochemical properties. Linear ester groups with varying chain lengths were introduced to systematically investigate the effects of the hydrophobic moiety distribution on lignin nanoparticle formation via solvent-shifting self-assembly. Results demonstrated that esterification-induced structural changes altered the balance of key noncovalent interactions (hydrogen bonding, π–π stacking, and hydrophobic interactions), which collectively governed the self-assembly process, with longer ester chains promoting compact particles with hydrophobic surfaces. By directly linking molecular-level modification of lignin to alterations in the inter- and intramolecular interactions driving the self-assembly of nanoparticles, this study provides a mechanistic framework for the rational design of lignin nanoparticles through controlled chemical modification, thereby expanding their application flexibility.