Gelatin nanoparticles (GNPs) of about 100 nm in diameter have been prepared from type B gelatin crosslinked with glutaraldehyde by the modified two-step desolvation process. The effects of the gelatin concentration in dispersions, desolvation solvent (acetone), and the amounts of the cross-linker (glutaraldehyde) have been investigated. The results show that the size and dispersion of the GNPs can be decreased by reducing the content of gelatin in the second dispersed solution before crosslinking. Moreover, the residual acetone in the as-prepared dispersion must be removed completely, since it leads to the forming of larger and less stable particles. Using only 4.5% (w / w gelatin) glutaraldehyde in the 8 g/L (w / v solution) gelatin dispersion can produce stable GNPs with the diameter of 50 ~ 150 nm, a polydispersity index of about 1.4, and Zeta potential < -30 mV. Atomic force microscope (AFM) and transmission electron microscope (TEM) analysis shows that the nanoparticles are spherical morphology and heterogeneous spheres whose edge parts are looser than the cores.