AbstractThe aim of this study was to develop a sensitive lateral flow immunoassay (LFIA) for the rapid detection ofEscherichia coli(E. coli) O157:H7, a pathogen contributor to diseases and fatalities worldwide. Au nanoparticles with high stability, uniform size, and shape were synthesized and coated with heterobifunctional PEG polymer with carboxyl groups, and they were bioconjugated to be used as label in sandwich-LFIA. Then, a silver enhancement strategy was developed as an accessible, rapid, and cost-effective approach for signal amplification to reduce the limit of detection (LOD). The optimal results were achieved when a solution of silver nitrate and hydroquinone/citrate buffer was added to the strips for 4 min. This led to a decrease in the visual LOD from 2 × 106(CFU mL−1) to 2 × 103(CFU mL−1), resulting in a threefold improvement in sensitivity compared to the conventional LFIA system. The specificity of the system was evaluated by using non-target bacteria (E. coliBL21 andE. coliT515) and its reliability was determined by testing commercial food samples (milk, tap water, and orange juice), demonstrating its effectiveness for quickly detecting pathogenic bacteria in food products.Graphical AbstractSupplementary InformationThe online version contains supplementary material available at 10.1007/s00604-023-05834-8.