Herein we report a new separable detection system combining magnetic nanoparticle labels with giant magnetoresistance (GMR) first time developed for Escherichia coli (E. coli) O157H:H7 detection. The GMR sensor fabricated employing standard microfabrication technique, while double antibody sandwich immunoassay and streptavidin–biotin binding assay were employed for immobilizing and labeling E. coli O157H:H7 on Au-film coated glass wafer. The GMR-based immunosensor detected E. coli O157H:H7 antigens at different sample concentrations with a minimum detectable limit of 100CFU/mL, well accepted for early detection in clinical diagnosis and environmental monitoring. Compared with the recently reported GMR sensor based detection methods, this newly developed separable detection method possesses several advantages such as convenient manipulation, avoid being contaminated and damaged by chemical solution and reusability without cleaning. So it is of considerable interest towards the biomedical application based on known specific binding of target and labels.