Gene transfection using bacterial magnetic particles (BMPs)-polyethylenimine (PEI) has become increasingly prevalent; however, relatively little effort has been made to optimize the protocol for preparing these complexes with the aim of improving their transfection efficiency. Here, we report a procedure for constructing BMPs-PEI/DNA complexes that results in improved transfection efficiency, reduced cytotoxicity and shorter procedure times for both complex formation and transfection over current methods. BMPs-PEI/DNA complexes mixed using ultrasonication yielded beads that were 10.2% more efficient at transfecting HeLa cells than complexes made by mechanical vortexing. Phosphate-buffered saline (PBS) proved to be a superior solvent for BMPs-PEI/DNA and PEI/DNA complexes, and the transfection efficiencies in HeLa cells were 54.75% and 46.01%, respectively. Comparable levels of transfection were achieved after 10 min of incubation with low-dose BMPs-PEI/DNA complexes versus 4 h with standard PEI/DNA complexes. BMPs stored in PBS have an average transfection efficiency that is 5% greater than those stored in physiological salt solutions. Cell morphology and cytotoxicity analyses demonstrated that the biosynthesized BMPs lessened the cytotoxicity of PEI to cells. Our results provide an optimized protocol for BMPs-PEI/DNA complex construction and gene transfer in vitro.