Gold nanoparticle(GNP)based dynamic light scattering(DLS)assay has been widely used for sensitive detection of small analyte based on analyte binding-induced GNP aggregation.However,this new method for detection of larger biological objectives such as pathogenic bacteria has not been reported.Herein,for the first time we described a homogeneous GNP-based DLS immunoassay for the ultrasensitive detection of Listeria monocytogenes.Compared to small analyte,L.monocytogenes with large surface and more antigen epitopes can serve as carriers binding to GNP probes to form "GNP-coated bacteria" complexes.To achieve a better analytical performance,various parameters including the GNP concentration and size,the amount of monoclonal antibodies on the surface of GNP,and immunoreaction time were systematically investigated and optimized.Under the optimum conditions developed,the limit of detection(LOD)for L.monocytogenes reached as low as 3.5 × 101 CFU mL–1 in 0.01 M phosphate-buffered saline.Coupled with a large-volume immunomagnetic separation method,the LOD for spiked lettuce samples achieved 2.2 × 101 CFU g–1,which was one order of magnitude lower than the maximum limit imposed in Canada(100 CFU g–1).Moreover,the proposed method exhibited excellent discrimination against seventeen common pathogenic bacteria in foods.In brief,the GNP-based DLS immunoassay developed here has great potential in application for detection of large biological objectives.