Biodegradable cross-linked poly(beta-amino ester) (CLPAE) was synthesized by Michael addition of pentaerythritol triacrylate and N,N-dimethylethylenediamine and modified with aminohexanoic acid and lysine to CLPAE-Ahx and CLPAE-Lys, respectively, for a gene delivery system. They could self-assemble with plasmid DNA, forming nanosized polyplexes, and CLPAE-Ahx polyplex released plasmid DNA slowly during a week through stepwise degradation. The polymers showed minimal cytotoxicity on 293 cells due to their biodegradability and biocompatibility. Transfection efficiencies of CLPAE-Ahx and CLPAE-Lys were comparable to that of PEI in 293 cells and C2C12 cells. Additionally, high transfection of CLPAE-Ahx on primary rat aorta vascular smooth muscle cells (SMC) and primary mouse embryonic fibroblast cells (MEF) shows a potential for a gene delivery system on primary cells, restenosis treatment of human SMC, and MEF cell function research. In conclusion, CLPAE-Ahx could be used as a nontoxic and highly efficient gene delivery system.