Abstract: Objective: To establish a method for the determination of Gram-negative bacteria - Escherichia coli by Colis-Fe₃O₄-graphene quantum dots (GQDs) functionalized fluorescent probes. Methods: Amino modified Fe₃O₄ was prepared by co-precipitation method, and it was combined with GQDs containing carboxyl groups, and Colis-Fe₃O₄-GQDs complex was formed by colistin modification. With this complex as fluorescent probe, different concentrations of Escherichia coli were detected. Results: The fluorescence quenching effect of Colis-Fe₃O₄-GQDs complex was good with Escherichia coli, and the fluorescence quenching of Colis-Fe₃O₄-GQDs complex was gradually enhanced with the increasing concentration of the strain. The linear detection range of this method was 1×10²~10×10² CFU/mL, the coefficient of determination was 0.999, and the detection limit was 0.36×10² CFU/mL. Conclusion: The colis-Fe₃O₄-GQDs biosensor is simple and efficient, with good selectivity and sensitivity, which provides a new method for the detection of Gram-negative bacteria, and has a broad application prospect in the detection of bacteria.