Lignin colloidal spheres (LCSs) are promising biomaterials for application in drug storage and delivery, pollutant adsorption, and ultraviolet protection due to their biocompatibility, amphiphilicity, and conjugated structure. However, wide size distribution of LCSs greatly limits their performances, especially in many precise and advanced applications. Herein, the fabrication of monodispersed LCSs with tailorable sizes ranging from the nanoscale to microscale is reported. Lignin raw materials are first fractionated by solvent extraction, and then the lignin fraction is used to fabricate monodispersed LCSs by solvent/antisolvent self‐assembly. The underlying mechanism for the formation of monodispersed LCS is primarily ascribed to the improved homogeneity of long‐range intermolecular forces, especially the electrostatic forces and hydrophobic forces, between lignin molecules. Moreover, by manipulating the short‐range order of LCSs, an innovative application of lignin as bio‐photonic materials with tunable structural colorations (e.g., red, green, or blue) is demonstrated. This work not only provides deep insight and an effective strategy to eliminate the serious inhomogeneity of LCSs, but also makes lignin resources have great potential as biodegradable and biocompatible photonic materials in diverse advanced optical application fields such as photonic devices, anti‐counterfeiting labels, and structural color pigments.