Polyanhydrides are considered as important biomaterials for use in drug delivery systems, due to their biodegradability, biocompatibility and appropriate release kinetics of active substances. They can be obtained by melt polycondensation of compounds containing two carboxylic groups, e.g. disuccinate betulin. Disuccinate betulin exhibits biological activity, including anticancer and antiviral one, while being non-toxic to normal cells. Nanoparticles prepared from betulin-based polyanhydrides may have significant application in drug delivery systems. The aim of this study was to obtain polymeric nanoparticles from polyanhydride based on betulin disuccinate and to evaluate the influence of the preparation conditions (homogenization time, type and concentration of surfactant, concentration of the polymer solution) on the shape, morphology and size of the particles. Nanoparticles were obtained by two methods: by nanoprecipitation and by emulsion solvent evaporation (ESE) method. Both methods allow to obtain particles with a diameter of less than 1 µm. ESE method provided regularly spherical particles, while among the particles obtained from the nanoprecipitation method, many irregular ones were observed. In ESE method, the size of the particles depended on the type and concentration of surfactant (in the water phase) and the polymer concentration (in the organic phase). The best results were obtained with ionic surfactants, however, the use of such compounds may accelerate the degradation process of polymers. In nanoprecipitation method, the ratio of solvent (methylene chloride) to non-solvent (hexane) has a significant influence on the particle size. The smallest particles were obtained with a solvent to non-solvent ratio of 1 : 150.