BACKGROUND Escherichia coli O157:H7 is one of the major foodborne bacterial pathogens and also a biodefense agent. To ensure food safety and public health, it is very important to develop rapid methods for E. coli O157:H7 detection. In this study, we designed a nanoparticle-labeled biosensor for the rapid detection of E. coli O157:H7 in broth. RESULTS Magnetic nanoparticles (MNPs) were conjugated with monoclonal antibodies (Abs) to separate target E. coli O157:H7 cells from broth samples. Gold nanoparticles (AuNPs) were conjugated with polyclonal Abs, and were then introduced to the MNP-target complex to form a sandwich MNP-target-AuNP. By measuring the amount of AuNPs through an electrochemical method, the presence and the amount of the target bacteria were determined. Results showed a sensitivity of 10(1) colony forming units per milliliter (cfu/ml) with a linear range of 10(1)-10(6) cfu/ml. CONCLUSIONS Compared to conventional culture plating methods, the biosensor reduced the detection time from 2 to 4 days to less than 1 hour with a simple target extraction method. The AuNP-labeled biosensor has potential applications in the rapid detection of infectious agents for public health, biodefense, and food/water safety.