A homogeneous fluorescence quenching immunoassay is described for simultaneous separation and detection of aflatoxin M (AFM) in milk. The novel assay relies on monoclonal antibody (mAb) functionalized FeO decorated reduced-graphene oxide (rGO-FeO-mAb) as both capture probe and energy acceptor, combined with tetramethylrhodamine cadaverine-labeled aflatoxin B (AFB-TRCA) as the energy donor. In the assay, AFB-TRCA binds to rGO-FeO-mAb in the absence of AFM, quenching the fluorescence of TRCA by resonance energy transfer. Significantly, the immunoassay integrates sample preparation and detection into a single step, by using magnetic graphene composites to avoid washing and centrifugation steps, and the assay can be completed within 10 min. Under optimized conditions, the visual and quantitative detection limits of the assay for AFM were 50 and 3.8 ng L, respectively, which were significantly lower than those obtained by fluorescence polarization immunoassay using the same immunoreagents. Owing to its operation and highly sensitivity, the proposed assay provides a powerful tool for the detection of AFM.