Polysaccharides and other cationic polymers have recently been used in pharmaceutical research and industry for their properties to control the release of antibiotics, DNA, proteins, peptide drugs or vaccines, and they have also been extensively studied as non-viral DNA carriers for gene delivery and therapy. Among them, chitosan is the most used since it can promote long-term release of incorporated drugs. This work is focused on the preparation of chitosan and chitosan/DNA nanospheres by using a novel and simple osmosis-based method, recently patented. The morphology of chitosan/DNA particles is spherical (as observed by scanning electron microscopy, SEM) and the nanospheres' average diameter is 38 ± 4 nm (obtained by dynamic light scattering, DLS). With this method, DNA is incorporated with high yield (up to 30%) and the release process is gradual and prolonged in time. The novelty of the reported method resides in the general applicability to various synthetic or natural biopolymers. Solvent, temperature and membrane cut-off are the physicochemical parameters that one is able to use to control the overall osmotic process, leading to several nanostructured systems with different size and shape that may be used in several biotechnological applications.