Various kinds of inorganic nanoparticles have been used as non-viral gene carriers. Two fundamental roles of gene carriers are to bind the DNA molecules and to protect them from enzymatic attack after internalization into the cells. Therefore, all nanoparticles that are used as gene carriers must be functionalized. Lately, magnetic gene carriers incorporating PEI have been adopted to improve DNA transfection efficiency. Researchers have used PEI-coated MNPs for DNA entrapment, and they have found that this complex was not able to achieve an efficient DNA transfection, but needed an extra free PEI to deliver the DNA to the cell nucleus. In this study, magnetic gene carriers with small sizes and surface modifications were prepared to explore the magnetofection process. Different methods for PEI immobilization on smaller MNPs were adopted to compare DNA binding abilities, transfection and transient gene expression efficiencies. Finally, the magnetofection process was studied with confocal microscopy and flow cytometry. These results provide details regarding the mechanism of DNA magnetofection, which has not been yet fully understood.