Infections from invasive Listeria monocytogenes (L. monocytogenes) frequently occur in food and can cause high morbidity and death. Thus, the sensitive, specific, and rapid detection of L. monocytogenes is critical for ensuring food safety and public health. Herein, a fluorescence immunoassay for trace L. monocytogenes detection was designed based on guinea pig antibody-functionalized magnetic nanoparticles (Fe3O4 NPs/pAb1) and rabbit antibody-anchored CdZnTe quantum dots (CdZnTe QDs/pAb2). Because of the antibody-directed magnetic separation and long-wave fluorescent emission for CdZnTe QD indication, the constructed immunoassay strategy presented excellent anti-interference performance toward a biological matrix. The immunosensor exhibited a wide detection range of 1 to 109 CFU mL-1 for L. monocytogenes and a low limit of detection (LOD) of 1 CFU mL-1, achieving an exceptionally sensitive detection of trace L. monocytogenes. Meanwhile, the immunosensor showed good specificity and had a short time-consumption of 60 min to realize the accurate determination of trace Listeria monocytogenes in spiked tap water and pasteurized milk samples.