Low transfection efficiency and inactivation by serum are the major drawbacks for cationic polymers when used as non-viral gene vectors. Here, a series of N-succinyl-chitosan-graft-polyethylenimine (NSC-g-PEI) copolymers with different compositions were synthesized through grafting low molecular weight PEI (800 Da) to N-succinyl-chitosan. An agarose gel electrophoresisassay showed NSC-g-PEIs had good binding capability with DNA and the particle size of the NSC-g-PEI–DNA complexes was between 150 to 300 nm as determined by a Zeta sizer. In vitro transfection of NSC-g-PEI–DNA complexes for 293T, HeLa and CHO cells was investigated. It was found that the transfection efficiency of NSC-g-PEI–DNA complexes was higher than that of DNA combined PEI (25 kDa) and the transfection efficiency increased with the increasing GD of PEI. More importantly, the NSC-g-PEI–DNA complexes were stable and the transfection efficiency was not affected obviously in the presence of serum with different concentrations. In addition, NSC-g-PEIs had a lower cytotoxicity than PEI (25 kDa) and the toxicity increased with increasing GD of PEI. The NSC-g-PEI copolymers will have a good potential as efficient non-viral gene vectors in the presence of serum.