Lignin has recently attracted much attention due to their renewable nature. Here we focused on a simple self-assembly method for fabricating size and shape uniform enzymatic hydrolysis lignin (EHL) nanospheres, without chemical modification of lignin. EHL was dissolved in tetrahydrofuran at different initial concentrations and subsequently self-assembly with adding water under magnetic stirring for fabricating the nanospheres. The self-assembled structure, process parameters and formation mechanism of the nanospheres Were investigated by transmission electron microscopy (TEM), scanning electron microscopy (SEM), dynamic light scattering (DLS), fourier transform infrared spectroscopy (FTIR), and UV-vis absorption spectra. Results showed that the nanospheres were formed through a layer-by-layer self-assembly approach from inside to outside based on pi-pi interactions, which enabled the formation of nanospheres in the size range of 190-590 nm. Increasing the pre -dropping EHL concentration resulted in an increase of the average diameter and yield of the nanospheres. The nanospheres have good stability, and their average diameters had no significant change after 30 days. The chemical structural features of the nanospheres had not produced a significant change in the preparation process. High preparation temperature brought about the formation of the gaps at the surface of the nanospheres due to the effect of volatile speed of solvent. Moreover, the average diameter of the nanospheres decreased with an increase of stirring rate or the dropping speed of water. The proposed EHL nanospheres are eco-friendly, cost-effective and therefore a promising candidate for biomass based carrier. (C) 2017 Elsevier B.V. All rights reserved.