Chitosans have been proposed as biocompatible alternative cationic polymers that are suitable for non-viral delivery. However, the transfection efficiency of chitosan-DNA nanoparticles is still very low. To improve transfection efficiency, we prepared chitosan-graft-polyethylenimine (CHI-g-PEI) copolymer by an imine reaction between periodate-oxidized chitosan and polyethylenimine (PEI). The molecular weight and composition of the CHI-g-PEI copolymer were characterized, using multi-angle laser scattering (GPC-MALS) and (1)H nuclear magnetic resonance ((1)H NMR), respectively. The copolymer was complexed with plasmid DNA (pDNA) in various copolymer/DNA (N/P) charge ratios, and the complex was characterized. CHI-g-PEI showed good DNA binding ability and high protection of DNA from nuclease attack. Also, with an increase in charge ratio, the sizes of the CHI-g-PEI/DNA complex showed a tendency to decrease, whereas the zeta potential of the complex showed an increase. The CHI-g-PEI copolymer had low cytotoxicity, compared to PEI 25K from cytotoxicity assays. At high N/P ratios, the CHI-g-PEI/DNA complex showed higher transfection efficiency than PEI 25K in HeLa, 293T and HepG2 cell lines. Our results indicate that the CHI-g-PEI copolymer has potential as a gene carrier in vitro.