Listeria monocytogenes continues to be a major foodborne pathogen that causes food poisoning, and sometimes death, among immunosuppressed people and abortion among pregnant women. In this study, magnetic nanoparticles with a diameter of 30 nm were functionalized with anti-L. monocytogenes antibodies via biotin-streptavidin bonds to become immunomagnetic nanoparticles (IMNPs) to capture L. monocytogenes in a sample during a 2-h immunoreaction. A magnetic separator was used to collect and hold the IMNPs-L. monocytogenes complex while the supernatants were removed. After the washing step, the nanoparticle-L. monocytogenes complex was separated from the sample and injected into a microfluidic chip. The impedance change caused by L. monocytogenes was measured by an impedance analyzer through the interdigitated microelectrode in the microfluidic chip. For L. monocytogenes in phosphate-buffered saline solution, up to 75% of the cells in the sample could be separated, and as few as three to five cells in the microfluidic chip could be detected, which is equivalent to 10(3) CFU/ml of cells in the original sample. The detection of L. monocytogenes was not interfered with by other major foodborne bacteria, including E. coli O157:H7, E. coli K-12, L. innocua, Salmonella Typhimurium, and Staphylococcus aureus. A linear correlation (R(2) = 0.86) was found between the impedance change and the number of L. monocytogenes in a range of 10(3) to 10(7) CFU/ml. Equivalent circuit analysis indicated that the impedance change was mainly due to the decrease in medium resistance when the IMNPs-L. monocytogenes complexes existed in mannitol solution. Finally, the immunosensor was evaluated with food sample tests; the results showed that, without preenrichment and labeling, 10(4) and 10(5) CFU/ml L. monocytogenes in lettuce, milk, and ground beef samples could be detected in 3 h.