Polyamidoamine-polyethylene glycol (PAMAM-PEG) copolymers were synthesized using IPDI as coupling reagent by two-step method.  The copolymers were characterized by IR spectrum and ¹H NMR spectrum, and the PEG conjugating ratios of the copolymers were calculated equal to 10% and 30% separately.  MTT assay indicated that after PEGylation a lower cytotoxicity of the copolymers could be found, and with increasing PEG conjugating ratio the cytotoxicity decreased obviously.  Agarose gel retardation assay demonstrated that PAMAM-PEG copolymers could be combined with DNA and PAMAM-PEG/DNA complexes were prepared by self-assembly.  DLS measurement showed that when N/P≥50, the particle size of copolymer/ gene complexes was in a range of 150−200 nm, and the zeta potential was in a range of 10−25 mV.  In vitro gene transfection illustrated that when N/P≤50, the gene transfection efficiency of PAMAM-PEG copolymers was a little less than that of PAMAM-G5, but the transfection efficiency can be raised by increasing N/P ratio or transfection time.  Considering both cytotoxicity and transfection efficiency aspects PAMAM-PEG-13 was more effect than PAMAM-PEG-39 in PEGylation.