Low transfection efficiency is always an issue when cationic polymers are used as a nonviral gene vector in the physiological condition, especially in the presence of proteins. A cationic magnetic nanoparticle (MNP) may be an alternative to solve this problem because a magnetic field can help to attract the MNP and internalize it into cells. The aim of this study was to determine the potency of polyethylenimine (PEI)-decorated MNPs for efficiently complexing and delivering plasmid DNA in vitro with the help of a magnetic field. PEI is associated with poly(acrylic acid)-bound superparamagnetic iron oxide (PAAIO) through electrostatic interactions (PEI-PAAIO). PEI-PAAIO formed stable polyplexes with pDNA in the presence and absence of 10% fetal bovine serum (FBS) and could be used for magnetofection. The effect of a static magnetic field on the cytotoxicity, cellular uptake, and transfection efficiency of PEI-PAAIO/pDNA was evaluated with and without 10% FBS. Magnetofection efficacy in HEK 293T cells and U87 cells containing 10% FBS was significantly improved in the presence of an external magnetic field. The amount of internalized iron was quantitatively measured using an inductively coupled plasma-optical emission spectrometer and directly visualized using Prussian blue staining. The internalized pDNA was visualized using a confocal laser scanning microscope.