We have developed a magnetic separation-based immunocolorimetric assay to detect sesame allergens. Sesame monoclonal antibody (Ab) was modified onto gold nanoparticles (AuNPs) to create signal probes (AuNPs–Ab), and sesame allergens (SA) were attached to carboxyl-functionalized magnetic polystyrene microspheres (MPMs) to act as capture probes (MPMs–SA). Based on the competition format, the capture probes competed with the sesame allergens in the sample to bind the corresponding signal probes. When sesame allergens were present, two immune complexes (AuNPs–Ab@MPMs–SA and AuNPs–Ab@SA) were formed. The immune complex AuNPs–Ab@SA was used to quantify the sesame allergens in the sample. This immunoassay had a detection linear range from 50 to 800 μg L⁻¹ with a limit of detection (LOD) of 45.53 μg L⁻¹. Based on the optimized conditions, the recovery of sesame allergens in bread, biscuit, almond beverage, and energy bar samples was between 82.50% and 116.67%. The LODs for the bread, biscuit, almond beverage, and energy bar samples were 0.36, 0.36, 0.27, and 0.55 mg kg⁻¹, respectively.