New glycidyl-based (epoxide-based) poly(aminoester) (EPAE) containing hydroxyl and amino groups in the backbone and side chain was synthesized. EPAE self-assembled readily with the plasmid DNA(pCMV-βgal) in HEPES buffer and was characterized by dynamic light scattering, Zeta-potential, fluorescence images, and XTT cell viability assays. To evaluate the effect of molecular weight of EPAE system on transfection, EPAE polymers with three different molecular weights (EPAE22k, EPAE18k, and EPAE8k) were also prepared. This study found that all EPAE polymers were able to bind plasmid DNA and yielded positively charged complexes with a nano-sized particles (200 nm). The EPAE22k/DNA and EPAE18k/DNA complexes were able to transfect COS-7 cell in vitro with higher transfection efficiency than other EPAE8k/DNA. These results demonstrated that molecular weight of EPAE system had a significant effect on transferring ability. Examination of the cytotoxicity of PEI25k and EPAEs system revealed that EPAEs system had lower cytotoxicity. In this article, EPAEs seemed to be a novel cationic poly(aminoester) for gene delivery and an interesting candidate for further study.