In this research, a magnetophoretic separation device was designed, fabricated, and tested using magnetic nanoparticles (MNPs) for more efficiently separating target foodborne pathogens. This magnetophoretic separation device consisted of a rectangular channel with a steel rod on its top, both of which were immersed in a homogenous magnetic field generated by two permanent magnets. A sample containing foodborne pathogens was incubated with biotin conjugated antibody (Ab) and then streptavidin-coated MNPs with a diameter of 30 nm to form MNPs-Ab-cell complexes, the magnetic cells. The magnetized steel rod concentrated the magnetic field and generated a high magnetic field gradient, which could separate the magnetic cells from a background fluid without need of repeated washing. First, a mathematical model was developed in order to understand and predict the performance of the separation device. Secondly, the magnetophoretic separation device was experimentally tested using Escherichia coli O157:H7 as a model bacterium. The results showed that 97.5% of the E. coli O157:H7 cells (4.4 × 103 CFU mL-1) could be separated from a phosphate buffered saline (PBS) solution at a flow rate of 14.4 µL min-1. In addition, a separation efficiency of 85.6% was achieved during the separation of E. coli O157:H7 from a mixture of E. coli O157:H7 (4.6 × 103 CFU mL-1) and Listeria innocua (5.7 × 103 CFU mL-1) in a PBS solution. The magnetophoretic separation device has great potential for broad applications in the preparation or pretreatment of a large volume of food, agricultural, and environmental samples in conjunction with a rapid biodetection method, such as biosensors, PCR, or ELISA, to more sensitively detect bacterial pathogens.