Gold nanoparticles (GNPs) conjugated with low molecular weight polyethylenimine (PEI 800 Da) were synthesized, and their characteristics as gene transfection vectors were investigated. The polyethylenimine conjugated GNPs (GNP-PEI800s) can retard plasmid DNA completely at N/P ratios above 4 in electrophoresis on agarose gel, and they also render effective protection of DNA from attack by DNase. TEM imaging revealed that GNP-PEI800s with higher PEI grafting density resulted in more compact and smaller complexes with plasmid DNA, compared to those obtained with lower grafting density ones. These complexes showed high efficiency in gene delivery in monkey kidney cells in vitro. In the absence of serum, GNP-PEI800s can transfect pGL-3 to COS-7 cells 3 to 4 orders more efficient than unmodified PEI800, reaching almost the same magnitude of PEI 25 kDa. More importantly, in contrast to the dramatically lowered efficiency of high molecular weight PEIs such as PEI 25 kDa in the presence of serum, the efficiency of GNP-PEI800s can be retained or even enhanced in serum-containing media. GNP-PEI800 1.3 exhibited transfection efficiency exceeding 60-fold that of PEI 25 kDa in 10% serum medium. All GNP-PEI800s exhibit mild cytotoxicity in comparison with that of PEI 25 kDa.