A novel anionic PEG derivative with a wheat-like structure, PEG-poly(AGE-Suc), was synthesized. The spikelet part of this polymer consisting of 9.3 pairs of carboxylic acid side chains was conjugated at one end of the PEG chain. The neutral linear PEG(1580) was designed as the stalk part to improve the biocompatibility of the vectors. The obtained polymer PEG-poly(AGE-Suc) was further modified by folate (FA) at the distal end to achieve the cell-specific targeting. It was confirmed that the negatively charged FA-PEG-poly(AGE-Suc) could coat the positively charged PEI 25K/DNA complex and form a ternary complex by electrostatic interaction. The addition of FA-PEG-poly(AGE-Suc) could change the positive charge of PEI 25K/DNA complexes to negative with no influence on the diameter. The ternary complex with the coat of FA-PEG-poly(AGE-Suc) could effectively condense DNA and protect it from degradation by DNase I. The nonspecific interaction between PEI 25K/DNA complexes and blood components was also significantly reduced by the addition of anionic PEG. The ternary complex PEI 25K/DNA/FA-PEG-poly(AGE-Suc) exhibited a 12-fold higher transfection efficiency on 293T cells compared to PEI 25K/DNA in the serum-containing medium. The competitive folate inhibition assay demonstrated that the higher transfection efficiency of PEI 25K/DNA/FA-PEG-poly(AGE-Suc) was attributed to the folate molecule conjugated to the distal end of FA-PEG-poly(AGE-Suc). The ternary complex PEI 25k/DNA/FA-PEG-poly(AGE-Suc) with low side effects and high transfection efficiency may be a novel effective gene delivery system.