This paper described that Fe3O4 nanoparticles were encapsulated with methyl methacrylate (MMA) using linolenic acid (LA) as a crosslinking agent, after which the resulting polymethyl methacrylate (PMMA) embed Fe3O4 nanoparticles (PMMA/Fe3O4) were coated with silica, forming SiO2/ (PMMA/Fe3O4) core-shell structure particles. Then these magnetic nanoparticles (MNPs) were applied in the developed system of chemiluminescent magnetic enzyme-linked immunoassay. E. coli O157:H7 was sandwiched between rabbits anti-E. coli O157:H7 polyclonal antibody-coated magnetite nanoparticles (immunomagnetic nanoparticles or IMNPs) and mouse anti-E. coli O157:H7 monoclonal antibody (E. coli O157-McAb). Commercial alkaline phosphatase conjugated horse anti-mouse immunoglobulin (ALP-Ab) was used to bond with the monoclonal antibody, finally the chemiluminescent signals were detected by adding 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD) which was the substrate reagent of ALP. The specificity and sensitivity of this system for detecting E. coli O157:H7 were researched. The results indicated that this method was of good specificity when using E. coli Top 10F' and Vibrio cholera as negative controls. The detection limit was 10(3) cells mL(-1) when the antigen solution was 1 mL, and the procedure duration was about 3 h.