To improve the transfection efficiency of poly(urethane-co-ester) and the cytotoxicity of PEI25k with DNA, we synthesized a new poly(urethane-co-ester), PUE, bearing ester linkages and amino groups in the backbone and urethane linkages in the side-chain, and then prepared a binary mixture, PUE-PEI25k, using a physical blending method. The structure of PUE was confirmed by FT-IR and NMR spectra. Both poly(urethane-co-ester), PUE, and binary mixture PUE-PEI25k, readily self-assembled with plasmid DNA (pCMV-βgal) in a HEPES buffer, were characterized by dynamic light scattering. The results revealed that PUE and PUE-PEI25k were able to self-assemble plasmid DNA into PUE/DNA and PUE-PEI25k/DNA nano-complexes small enough to enter a cell through endocytosis. Titration studies were performed to determine the buffering capacities of PUE and PUE-PEI25k. The COS-7 cell viability in the presence of PEI25k, PUE and PUE-PEI25k was studied. At low mass ratio of PUE/PEI25k (150:1), it was found that the PUE-PEI25k/DNA complexes were able to transfect COS-7 cells in vitro with a high efficiency comparable to a well-known gene carrier, PEI25k/DNA. The results indicate that the binary mixture PUE-PEI25k is an attractive cationic carrier for gene delivery and an interesting candidate for further study.