Ferroferric oxide (Fe₃O₄) magnetic nanoparticles are widely used as passive targeting carriers in gene therapy, due to their simple preparation, targeting under external magnetic field and easy surface grafting. This study synthesized oil phase Fe₃O₄ nanoparticles with controllable particle sizes in the range from 4 to 9 nm by regulating the accumulation growth time in the solvothermal method. Then, meso-2, 3-dimercaptosuccinic (DMSA) was employed to double exchange oleic acid molecules on its surface to provide good water dispersibility. Finally, Fe₃O₄-DMSA-PEI magnetic nanoparticles were obtained by grafting branched polyethylenimine (PEI) onto Fe₃O₄-DMSA surface through amidization reaction. The results demonstrate that the Fe₃O₄-DMSA-PEI magnetic nanoparticles have a surface Zeta potential of (52.50 ± 1.94) mV, remaining a certain degree of superparamagnetism (14.48 emu/g, 1 emu/g=1 A∙m²/kg). When the mass ratio of Fe₃O₄-DMSA-PEI magnetic nanoparticles to plasmid DNA is 15 : 1, it can completely block DNA and its loading capacity is as high as 6.67%. The Fe₃O₄-DMSA-PEI magnetic nanoparticles prepared in this study have a certain gene delivery ability and are expected to be used as gene carriers in the field of gene transfection.