Listeria monocytogenes is an intracellular foodborne pathogen that has been associated with severe human illnesses. Various rapid detection methods have been developed for the specific detection of this pathogen. In the present study, a real-time quantitative polymerase chain reaction (PCR) assay targeting iap, a gene encoding extracellular protein p60, was developed for L. monocytogenes. The PCR efficiency is above 85% and the limit of detection (LOD) is 30 copies of genome per reaction for all strains tested. The assay exhibited 100% inclusivity and exclusivity rates. The detection of L. monocytogenes in five food matrices, whole milk, soft cheese, turkey deli meat, smoked salmon, and alfalfa sprouts, was evaluated with and without enrichment. Without enrichment, the LOD for all food matrices were 4×10(3) CFU/mL food enrichment mix for whole milk and 4×10(4) CFU/mL for all other foods. With 24 h incubation in Buffered Listeria Enrichment Broth, the LOD was 3 CFU/25 g food for whole milk, turkey deli meat, and smoked salmon and 9 CFU/25 g food for soft cheese and alfalfa sprouts. With 48 h incubation, the LOD was 3 CFU/25 g food for all matrices. This quantitative PCR appears to be a promising alternative for rapid detection of L. monocytogenes in select foods.