Rapid, simple, and sensitive detection of pathogen bacteria is a highly topical research area due to increasingly concerning of food safety and public health. Surface-enhanced Raman spectroscopy (SERS) is a promising and attractive technique offering fast, sensitive, comparatively low-cost, and in-suit detection of pathogenic bacteria. However, this technique requires the preparation step for reducing the noise derived from heterogeneous matrixes of food sample. Immunomagnetic separation (IMS) is widely used technique enabling separation and concentration of the target analyte. It can be used not only laboratory scale but also field diagnosis easily. Here, we synthesized gold-shelled starch magnetic microparticles (GS@SMMPs) for effective separation and concentration of Escherichia coli O157:H7, which were subsequently subjected to SERS integrated with gold-coated 3D-well substrate for bacterial detection in aqueous solution. GS@SMMPs were labelled by Anti-E. coli O157 monoclonal antibody through gold binding protein and staphylococcal protein G (GBP-SPG) fusion protein. In IMS experiment, the immuno-GS@SMMPs showed high capture efficiency over 90% to E. coli O157:H7, which resulted in 10 times decrease in detection limit in PCR assay. Through SERS assay, E. coli O157:H7 concentrated by immuno-GS@SMMPs were successfully detected even at an extremely low concentration of 101 CFU/ml the subjected to SERS. Moreover, by using sandwich method using SERS reporter consisting of GBP-SPG, we found that E. coli O157:H7 were able to be detected by SERS quantitatively through measuring the SERS intensity of GBP-SPG. This novel strategy combining SERS and IMS could be meaningful for extending the application in SERS for in-suit sensitive detection of pathogenic bacteria.