In this study, we describe the development of an automated immunomagnetic separation device combined with real-time polymerase chain reaction (PCR) for detecting foodborne bacteria. Immunomagnetic separation (IMS) is a well-known method for the separation and concentration of target bacteria from a large volume of food samples. Magnetic beads functionalized with an antibody provide selectivity for target bacteria such as Escherichia coli O157:H7. Moreover, compared to conventional methods, real-time PCR enables high-sensitivity detection of target bacteria. The method proposed in this study involves three steps: (1) pre-enrichment, (2) automated IMS and concentration of target bacteria, and (3) detection of target bacteria by real-time PCR. Using food samples with a working sample volume as large as 250 mL, the whole process only requires 3 h. As a result, target bacteria in the range of 101−102 colony-forming units per mg or g of sample can be detected in food samples, such as milk, ground beef, and cabbage, by using the proposed approach. We anticipate that the automated IMS system combined with real-time PCR will contribute to the development of a fully automated system for detecting foodborne bacteria and serve as a multi-tester for a variety of bacterial strains in the capacity of a sample-to-answer device in the near future.