Sulfonylureas, a family of anti-diabetic drugs widely used in the clinical treatment of type 2 diabetes, have recently emerged as an illegal adulterant in functional foods, to enhance the claimed anti-diabetic activity. To establish a screening assay method against their adulteration, with the aid of molecular simulation of hapten, two antibodies were raised and complementarily used to enhance the broad-specificity of an enzyme-linked immunosorbent assay (ELISA), which demonstrated simultaneous detection capability to 6 sulfonylureas; the detection limits ranged from 0.02 to 1.0 ng/mL, and recoveries were between 78.3% to 104.5%. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) confirmed the reliability of the proposed ELISA, based on real samples. These results suggest that the proposed ELISA could be an ideal method for screening to monitor for illicit adulteration of sulfonylureas in functional pill products.