Herein, Au nanocluster (AuNC)-embedded chitosan (CS) nanocapsules were prepared and used as a novel signal amplification system for the sensitive fluorescence immunoassay of Escherichia coli O157:H7 (E. coli O157:H7). AuNCs were embedded in CS to form AuNCs@CS nanocapsules, where a large number of AuNCs were embedded in each nanocapsule. A rapid, ultrasensitive, and selective method for the fluorescence detection of E. coli O157:H7 was developed by coupling the nanocapsule-amplification strategy with immunological recognition and magnetic separation. Compared with a conventional immunoassay using AuNCs as labels, the fluorescence signals of the proposed immunoassay were greatly amplified, because much more fluorescent AuNCs are linked to each bacterial cell. As a result, a linear range was obtained from 3 to 700 CFU mL-1, with a detection limit of 1 CFU mL-1. This method was successfully used to detect E. coli O157:H7 in drinking water and milk samples.