Gene delivery using the force of a magnetic field is called magnetofection. The purpose of this study is the synthesis and characterization of magnetic iron oxide nanoparticles (Fe3O4) as the core of the transfer agent and to investigate the effect of alternating magnetic field on transfection efficiency. For this purpose, the first magnetic nanoparticles (MNP) were synthesized by coprecipitation method. The magnetic properties of the synthesized MNP were investigated by vibrating sample magnetometer (VSM), appearance characteristics, and zeta potential of the synthesized particles were evaluated using transmission electron microscopy (TEM) and dynamic light scattering (DLS). Then, using magnetic nanoparticles (MNP), polyethylene imine (PEI) and plasmid DNA containing luciferase reporter gene (pDNA), PEI-pDNA binary complex and MNPPEI-pDNA ternary complex were synthesized. The complexes were evaluated using DLS and gel retardation techniques. The results of DLS and gel retardation technique showed that the complexes have a suitable surface charge and polyethyleneimine is well joined to pDNA and neutralized its negative charge. Finaly, human breast cancer cell lines (MCF-7) and Hek293T cells were transfected by ternary complex in the presence of 50 Hz alternating magnetic field. Cell viability was measured using the MTT test. The obtained results showed that the transfection efficiency in the cells that were transfected with the ternary complex in the presence of alternating magnetic field increased significantly compared to the control group, without any additional toxicity (P ≤ 0.05)