A new two-step desolvation method for manufacturing gelatin nanoparticles was developed. After the first desolvation step, the low molecular gelatin fractions present in the supernatant were removed by decanting. The high molecular fractions present in the sediment were redesolved and then desolvated again at pH 2.5 in the second step. The resulting particles can then be easily purified by centrifugation and redispersion. The different fractions obtained during the process were analysed by gel permeation chromatography (GPC). Based on these results, it can be concluded that the molecular weight of gelatin has a decisive influence on the stability of the manufactured gelatin nanoparticles. In addition, two fluorescent dyes (Texasred and fluoresceinamine) were coupled to the nanoparticles for cell uptake studies. The fluorescent nanoparticles showed a high uptake into monocytes/macrophages.