Acetylated lignin (ACL), obtained from the acetylation of alkali lignin (AL), has been found to self-assemble into ordered colloidal spheres in a THF/H2O media. This colloid formation causes the color of ACL to change from dark brown to light yellow. ACL molecules form colloidal spheres at critical water content (CWC) of 48 vol % for an initial concentration of ACL in THF of 0.4 mg/mL. The colloidization process is completed at a water content of 77.75 vol %. The colloidal spheres formed from the gradual hydrophobic aggregation of ACL were approximately 80 nm in size. The concentration of Fe3+ before and after colloid formation did not change significantly, which indicates that the whitening of ACL colloids is not influenced by metal ions, but is affected by the rearrangement of chromophores during self-assembly and the light reflectance of uniform particles. Self-assembly of ACL into colloidal spheres not only whitens the dark color of lignin but also provides a novel method to prepare ordered lignin-based nanomaterials.