To improve the cytotoxicity of PEI25k and the transfection efficiency of poly(β-amino ester) with DNA, we synthesized a poly(β-amino ester), PEDP, bearing ester linkages in the backbone and tertiary amines in the backbone and side chain and prepared a binary mixture, PEDP–PEI25k, using physical blending meyhod. Both poly(β-amino ester) PEDP and binary mixture PEDP–PEI25k, readily self-assembled with plasmid DNA (pCMV-β gal) in a HEPES buffer, were characterized by dynamic light scattering. The results reveal that PEDP–PEI25k was able to self-assemble plasmid DNA into PEDP–PEI25k/DNA nano-complexes small enough to enter a cell through endocytosis. Titration studies were performed to determine the buffering capacities of PEDP and PEDP–PEI25k. The COS-7 cell viabilities in the presence of PEDP and PEDP–PEI25k were studied. At low mass ratio of PEDP/PEI25k (1/1), it is found that the transfection curve of PEDP–PEI25k/DNA bearing a maximum peak is similar to that of PEI25k/DNA. In addition, the PEDP–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 binary mixture PEDP–PEI25k is an attractive cationic carrier for gene delivery and an interesting candidate for further study.