To improve the transfection efficiency of polycations with DNA, we synthesized poly(ester-co-urethane)(PEU-g-PEI800) with short chain PEI800 in the side chain, and poly(ester-co-urethane)(PEU) without short chain PEI800. Both PEU-g-PEI800 and PEU, readily self-assembled with plasmid DNA (pCMV-betagal) in a HEPES buffer, were characterized by dynamic light scattering and zeta-potential. The results reveal that PEU-g-PEI800 and PEU were able to self-assemble particles with DNA and yield nano-sized complexes (<200nm) with positive charge at N/P ratios of 20/1 and 120/1, respectively. The degradation studies indicate that the half-life of PEU-g-PEI800 and PEU in the HEPES buffer were 14 and 35h at pH 7.4, respectively. Titration studies were performed to determine the buffering capacities of the polymers. The COS-7 cell viabilities in the presence of PEU-g-PEI800/DNA, PEU/DNA, and PEI25k/DNA were studied. In addition, The PEU-g-PEI800/DNA complexes were able to transfect COS-7 cells in vitro with a high efficiency comparable to a well-known gene carrier PEI25k. The results indicate that PEU-g-PEI800 is an attractive cationic poly (ester-co-urethane) for gene delivery and an interesting candidate for further study.