ABSTRACT: There is an urgent need for a rapid and sensitive method for the detection of Escherichia coli O157:H7, a class of hazardous foodborne pathogens in food safety. The traditional ELISA, a dominant rapid detection technique, has the disadvantage of low test sensitivity due to the insufficient enzyme loading capacity. In this study, we successfully synthesized self-assembled Au/polydopamine (PDA)/horseradish peroxide (HRP) nanocomposites with high enzyme loading on the outer surface and in the inner space. The high catalytic activity of Au/PDA/HRP was maintained by virtue of its hyperbranched flexible structure. For E. coli O157:H7 detection in milk samples, the proposed immunoassay achieved a visual cut-off value of 103 cfu mL−1 and a low limit of detection of 2.8 × 102 cfu mL−1, which are 33 and 46 times more sensitive than the traditional ELISA, respectively. The tremendous advantages of high sensitivity, excellent specificity, and adequate recovery make it promising for monitoring various kinds of pathogenic bacteria in food safety with greater sensitivity.