A series of amphiphilic multi-armed PPn copolymers were prepared by ROP of Phe-NCA with PEI-25k as a macroinitiator. The particle size of the PPn/DNA complexes was about 100 nm and the zeta potentials were below 20 mV. An MTT assay demonstrated that all the PPn copolymers had lower cytotoxicity compared to PEI-25k. In vitro gene transfection studies were also conducted in HeLa, 293 and CT 26 cells. The optimal quantity of hydrophobic phenylalanine segments in PP80 led to higher transfection efficiency in various cell lines based on this study. The results indicate that PP80 was the best candidate for gene delivery among these PPn copolymers.