Kraft lignin (KL), a by-product in the pulping and papermaking industry, is an abundant, renewable resource. In this work, nanosphere formation of KL has been investigated via self-assembly induced by adding water to a KL/dioxane solution. The KL nanospheres were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (dynLS) and static light scattering (statLS). It was found that the nanoparticles have uniform nanospherical morphologies, including a small hollow cavity space in the interior. The π-π interactions between the aromatic rings of KL are demonstrated to be an important driving force in the self-assembly process. The KL nanosphere sizes can vary by hundreds of nanometers depending upon the preparation conditions. These newly obtained KL nanospheres have the advantages of biocompatibility, biodegradability, low toxicity, easy preparation, and low cost. Thus KL nanospheres are attractive for applications in life science, medicine, biology, food science, and agriculture. This approach presented here is an economically feasible and facile strategy for the sustainable utilization of kraft lignin.