Escherichia coli O157: H7 (E. coli O157: H7) is a foodborne pathogenic bacterium which can cause fever, diarrhoea and vomiting in humans. Thus, a rapid, simple, and specific bioprobe for pathogen detection in contaminated foods has been attracted more and more attention. In this work, the strong fluorescent amino-functionalized graphene quantum dots (af-GQDs) were prepared by hydrothermal method. The microtopographic height, surface morphology and spectroscopic properties of af-GQDs are characterized by the high resolution transmission electron microscope (HRTEM), atomic force microscope (AFM), UV-vis, fluorescence, Raman spectroscopic techniques. All the results showed that the af-GQDs can be effectively applied in the preparation of biocompatible immunofluorescence probe and in the detection of E. coli O157: H7. The minimum detection limit is 100 cfu/mL. It is a simple, rapid, sensitive, low-cost and easy to be popularized method, which provides a feasible way to monitor E. coli O157: H7 in food safety.