Gene therapy using polymers such as chitosan shows good biocompatibility, but low transfection efficiency. The mechanism of folic acid (FA) uptake by cells to promote targeting and internalization could improve transfection rates. The objective of this study was to synthesize and characterize FA-chitosan-DNA nanoparticles and evaluate their cytotoxicity in vitro. Chitosan-DNA and FA-Chitosan-DNA nanoparticles were prepared using reductive amidation and a complex coacervation process. The effect of charge ratio on the properties of these nanoparticles was monitored by laser scattering. DNA inclusion and integrity was evaluated by gel electrophoresis. Cell viability was illustrated with the MTT assay. Charge ratio (N/P) controlled the nanoparticles size and their zeta potential. Nanoparticles presented a mean size of 118 nm and 80% cellular viability compared to 30% cell viability using LipofectAMINE2000 controls. Gel electrophoresis showed intact DNA within the carriers. FA-nanoparticles have lower cytoxicity, good DNA condensation, positive zeta potential and particle size around 118 nm, which makes them a promising candidate as a non-viral gene vector.