Organic-inorganic hybrid silica nanoparticles (PM-1, PM-2 and PM-3) with positive surface charge and size below 200 nm were one-pot prepared via Michael addition between 3-methacryloxypropyl-trimethoxysilane (MPTMS) and polyethylenimine (PEI), followed by hydrolysis and polycondensation of siloxanes. The nanoparticles were characterized by FT-IR, element analysis and particle size analysis. The average sizes of the nanoparticles were 130-180 nm and the surface charge was about 40 mV. The acid-base titration showed that the nanoparticles had higher buffer capacity than PEI 25 kDa. The positively charged nanoparticles can condense negatively charged DNA to form complexes and completely retard the DNA mobility in agarose gel at a weight ratio of 5. The average sizes of PM-1/DNA and PM-2/DNA complexes were below 250 nm and the surface charge of the complexes was in the range of 30-40 mV at the weight ratio of 100. An in vitro transfection assay demonstrated that the transfection efficiencies of the nanoparticles were dependent on the PEI content, and PM-1 showed improved transfection efficiency compared with PEI 25 kDa in the presence of 10% serum. The intracellular trafficking assay of PM-1 nanoparticle/Cy3-labelled DNA complexes in COS-7 cells in the presence of 10% serum indicated that a large amount of complexes crossed the cell membrane and located in the cytoplasm and only a small amount of complexes entered into the cell nucleus after 24 h incubation. The uptake of PM-1 nanoparticle/DNA complexes by COS-7 cells in the presence of serum was higher than that of PEI/DNA complexes. In addition, the cytotoxicity of PM nanoparticles was significantly lower than that of PEI 25 kDa. The results indicate that the synthesized nanoparticles will show potential in nonviral gene delivery.