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A biosensing system, including an immunomagnetic separation (IMS), a flow injection and a bienzyme electrode, was developed for rapid detection of Escherichia coli O157:H7 in food samples. Samples inoculated with E. coli O157:H7 were mixed simultaneously with magnetic beads coated with anti-E. coli antibodies and alkaline phosphatase labeled anti-E. coli (APLAE) antibodies to form beads-E. coli-APLAE conjugates by antibodyantigen reaction. The conjugates were separated by a magnetic field and then incubated with phenyl phosphate to produce phenol. An amperometric tyrosinasehorseradish peroxidase biosensor in a flow injection system was used to detect the phenol concentration that is proportional to the cell number of E. coli O157:H7. The biosensor was evaluated using samples of chicken carcass wash water, ground beef and fresh-cut broccoli. This biosensor was able to detect as few as 6 10 1 cells/ml of E. coli O157:H7 under optimized conditions (1 mM MgCl2, 0.4 mg/ml APLAE, and 1 mM phenylphosphate in 25 mM Tris buffer solution pH 10.0). The total detection time from separating target bacteria with immunomagnetic beads to analyzing flow injection electrochemical detection was approximately 2 h.