As a powerful technology to enhance the efficiency of gene delivery, magnetofection has attracted considerable attention in the past decade. In this work, we introduced 6 generation of PAMAM dendrimer modified superparamagnetic nanoparticles (DMSPION-G6) to PEI/DNA polyplexes by a two-step process and enhanced the transfection efficiency of PEI with the help of a magnetic field. We prepared DMSPION-G6/DNA/PEI ternary magnetoplexes by precondensing DMSPION-G6 with DNA at a low mass ratio to yield DMSPION-G6/DNA magnetoplexes with negative surface charge, followed by further coating with branched PEI (25 kDa) via electrostatic interactions. We measured the transfection efficiencies of DMSPION-G6/DNA/PEI ternary magnetoplexes in COS-7, 293T and HeLa cells in the presence or absence of a magnetic field. Compared with PEI/DNA polyplexes, DMSPION-G6/DNA/PEI ternary magnetoplexes exhibited enhanced transfection efficiencies in all the three cell lines when a magnetic field was applied, especially in the presence of 10% FBS. Time-resolved and dose-resolved transfection indicated that high-level transgene expression was achievable with a relatively short incubation time and low DNA dose when magnetofection was employed. Further evidence from Prussian blue staining, quantification of cellular iron concentration and cellular uptake of Cy-3 labelled DNA demonstrated that the magnetic field could quickly gather the magnetoplexes to the surface of target cells and consequently enhanced the uptake of magnetoplexes by the cells. This represents a novel strategy for polycation-based in vitro gene delivery enhanced by a magnetic field.