Fairly monodispersed aminated gelatin nanoparticles (AGNPs, PDI ≈ 0.068) were prepared by a two-step desolvation method using amino-modified type B as raw material. The particle is positively charged (ζ ≈ 35 mV), with an average size of 240 nm. The effects of pH on the size and dispersity of AGNPs is investigated in the preparation process. It shows that the optimized reaction condition is pH = 4, which can endow the AGNPs with a smaller particle size (≈ 240 nm) and uniform distribution. The results from atomic force microscopy (AFM), scanning electron microscope (SEM) and transmission electron microscopy (TEM) showed that the AGNPs exhibited spherical morphology with a narrow particle size distribution. And compared with gelatin nanoparticles (GNPs), the rigid structure and homogeneity of AGNPs is enhanced. Moreover, the higher three-phase contact angle of AGNPs (θow = 67° ± 5°) than that of GNPs (θow = 31° ± 6°), as evaluated by surface wettability analysis, indicates the increased surface hydrophobicity and capability for the stabilization of O/W Pickering emulsion. Pickering emulsion stabilized by AGNPs can store at 4 ℃ for 9 months without stratification, possessing good storage stability.