Abstract: Gelatin nanopaticles about 100 nm in diameter were prepared by two-step single-desolvation improved, and the influence of the content of flocculant, pH value, desolvating and hardening times was investigated, respectively. The optimum conditions for the preparation are as follows: volume fraction of flocculant, 0.444; pH value, 3.8; desolvating time, 10 min; and hardening time, 25 min. The surface of gelatin particles is modified by adsorbing sodium poly(styrene sulfonate)(PSS), which made it negatively charged, and the amount of adsorbed PSS per gelatin nanoparticle determined by titrating in reverse is 8.23 mg. It may be provide the base of adsorbing medicines with positive charge.