The present paper describes the preparation and characterization of hydrophilic hyaluronan nanoparticles formed by cross-linking of hyaluronic acid (HA) with 2,2′(ethylenedioxy)bis(ethylamine) in the presence of water-soluble carbodiimide (CDI) in aqueous media. The particle size of cross-linked nanosystems was measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM), transmittance by UV–VIS spectrophotometry, gel permeation chromatography (GPC) and rheology to characterize the physico-chemical properties. The aqueous nanosystems prepared were stable, transparent or mildly opalescent with values of transmittance above 91%. It was observed, that a fraction of particles with size less than 20 nm, has been released during the purification by diafiltration. It was established that the hydrodynamic size of cross-linked HA nanoparticles can be controlled by variation of the reaction conditions such as concentration of HA, salt concentration of media, ratio of cross-linker and the final pH of the reaction mixture.