Hyperbranched poly(amido amine) (HPAMAM), which is structurally analogous to PAMAM dendrimers, has been proposed to be an effective agent for gene delivery. The facile synthesis of HPAMAM with scalable productivity by one-pot polymerization of monomers of methyl acrylate (MA) and diethylenetriamine (DETA) has been set up previously. In this study, the HPAMAM was further modified on the terminal amino groups with phenylalanine to various degrees (HPAMAM-PHE30, PHE45, PHE60). We showed that HPAMAM and HPAMAM-PHEs were all able to form complexes with plasmid DNA (pDNA) at various mass ratios. The cytotoxicity and transfection efficiencies of these polymers were evaluated in SMMC-7721 and COS-7 cell lines. The PHE modifications affected the cell transfection efficiency significantly. The HPAMAM-PHE60 was the most efficient, with transfection activities consistently higher than the commercial transfection reagent PEI. Our study demonstrated that HPAMAM-PHEs may be good new materials for gene delivery and other applications because of its large-scale availability. economical cost, and,low toxicity.