This study aimed to determine the optimal conditions for coupling immunomagnetic bead separation (IMS) with a dithiobissuccinimidyl propionate (DSP)-modified immunosensor and apply the IMS-coupled DSP-modified immunosensor to detect Listeria monocytogenes in chicken skin. Specific anti-Listeria polyclonal antibodies (pAbs) against three L. monocytogenes species were covalently immobilized on magnetic beads (MBs) to prepare immunomagnetic beads (IMBs). The optimal pAb concentration, incubation time, buffer type and pH, amount of MBs, and elution buffer for IMS were determined to be 1.0 mg/mL, 30 min, PBS buffer with a pH range from 6.5 to 7.5, 30 mu L, and citric acid (pH 3.0), respectively. The number of L. monocytogenes after IMS application was 6.0 log colony-forming unit (CFU)/g during the 15-h enrichment period, as compared to 7.1 log CFU/g without IMS application. IMS coupled with a DSP-modified immunosensor decreased the incubation time to 7.5 h with a limit of detection of 10(3) CFU/25 g chicken and improved the sensitivity of the immunosensor, exhibiting a correlation coefficient (R-2) of 0.95. Microscopic images of the immunosensor coupled with IMS revealed less binding of food particles on MBs than binding without IMS treatment. (c) 2014 The Electrochemical Society. All rights reserved.