A new method has been developed to improve the detection of norovirus (NoV) in complex fecal samples using nanomaterials-based immunoassays. The method involves using multifunctional trimetallic nanoparticles, known as Ag@Fe3O4@Au NPs. These nanoparticles consist of a core of silver (Ag) and a shell of iron oxide (Fe3O4) decorated with isolated gold (Au) nanoparticles. The nanoparticles have enhanced catalytic activity, making them an ideal nanocatalyst for reducing substrate molecules. The developed Ag@Fe3O4@Au NPs-based immunoassay achieved a limit of detection (LOD) of 1.9 pg/mL for norovirus-like particles (NoV-LP) and 6.97 fecal NoV copy number/mL. Achieving a LOD under 10 copy numbers is important for detailed protein analysis. The study also found that heat treatment of fecal suspension at 65°C was necessary to prevent the degradation of the target analyte for sensitive NoV detection. This work successfully combined multifunctional nanocatalysts for advanced immunoassays, which could contribute to developing nano-biosensing platforms.