We developed a Amylose magnetic beads (AMBs) based detection system for high efficient separation, concentration and detection of E. coli O157:H7 in real sample. AMBs were synthesized by amylosucrase from Deinococcus geothermalis (DgAS) with iron-oxide nanoparticle (NP). The design of amylose magnetic beads (AMBs) have studied by an enzymatic synthesis with optimized reaction condition such as substrate, sucrose, and iron-oxide NP. AMBs have specific feature. AMBs decorated with functional fusion protein, which consists in a maltose binding protein (MBP) and a streptococcal protein G (SPG). Amylose chains has maltose, thus MBP-SPG binds to the AMB. In addition, SPG specifically binds to the Fc part of antibody. That was used as a linker to immobilize antibody to the surface of AMBs. The resulting AMBs were efficiently separated and concentrated target bacteria, E. coli O157:H7. Concentrated sample is qualitatively analyzed by PCR. Our studies demonstrated that AMB-based PCR significantly reduced the limitation of detection as low as 10 1 CFU/mL, compared to that of conventional PCR. The principle of this system can be served as a high efficiency for detection method of any pathogenic bacteria. In addition, AMBs and MBP-SPG cross-linker protein developed in this study is expected to be applicable to the portable food based biological processing monitoring system.