Gene therapy is the treatment of genetic mutations and diseases by inserting therapeutic genetic material into diseased cells. The aim of this work is to evaluate the potential of magnetic iron oxide nanoparticles (MNPs) prepared from different synthesis techniques for use as non-viral vector in gene therapy. Two specimens of MNPs were prepared, one from the co-precipitation of iron (II) and iron (III) chlorides in an alkaline solution (CoP-MNPs), and the other from the flame spray pyrolysis of iron (III) acetylacetonate (FSP-MNPs). The MNPs were coated with 25 kDA polyethylenimine for conjugation with firefly reporter genes (pCMVLuc) and to improve the nanoparticles size dispersion. Gene expression in the cells was monitored by measuring the bioluminescence intensity. The MNPs uptake into human kidney cells (HEK293A) was visualised by Prussian Blue staining and quantified by ICP-OES. Up to 52-fold and 35-fold enhancement of gene expression were obtained with FSP-MNPs and CoP-MNPs, respectively, when the cells were placed under the influence of an external magnetic field for 1 hour (magnetofection). Dynamic light scattering measurements showed that the average size of the MNPs aggregates was reduced to less than 200 nm and remained stable for more than 14 days due to the steric effect of polyethylenimine. The stability and biocompatibility of the MNPs is pertinent to the use of the MNPs in vivo. Different MNPs specimen shows different gene delivery ability. Improved gene expression of FSP-MNPs compared to CoP-MNPs demonstrates the potential of using FSP-MNPs as a non-viral vector to enhance transfection efficiency.