A designed low aggregated magnetic polyethyleneimine/DNA (MPD-cc) complex showed efficient transfection in serum-containing medium for PEI-mediated gene transfection in vitro and in vivo, but the mechanism remains unclear. The present study provides an insight into the extracellular and intracellular fates of the magnetic gene complexes, evaluates their transfection efficiency and body distribution after systemic administration assisted by fluorescent imaging technology. The PEI cationic complexes in our study switched to be negatively charged in the serum-containing medium due to protein corona formation, and the complexes displayed negligible aggregation from transmission electron microscopy observation and dynamic light scattering analysis. However, the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that less protein was adsorbed on the magnetic gene complexes during a 10 min magnetofection compared with that associated with traditional polyethyleneimine/DNA (PD) complexes after a long-term (4 h) incubation. In terms of cellular uptake and internalization evaluation by flow cytometry, magnetofection with our designed MPD-cc complexes showed obvious superiority in the presence of serum, compared to traditional transfection (PD complexes). Moreover, confocal laser scanning microscopy revealed that after internalization, fewer MPD-cc with magnetofection were trapped in the endosome and more found in the nucleus compared to the PD and MPD-cc without the magnetic field. Putting these facts together, we conclude that magnetofection by the designed MPD-cc complexes facilitates many processes of transfection, including less protein adsorption, efficient cellular sedimentation and internalization, as well as endosomal escape and nuclear import. In the near infrared imaging study, it was observed that the accumulation of complexes in tumors by magnetic capture was enhanced in vivo. All of these improved in vitro and in vivo functions contributed to a 5-fold enhancement in magnetofection via intravenous delivery.