Mebendazole (MBZ), a synthetic benzimidazole, is most widely used for the treatment of intestinal helminthiasis. In the present study, a hapten mimicking the MBZ structure was designed by introducing propanoic acid and coupling to carrier proteins by the active ester method to immunize New Zealand rabbits. A sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the analysis of MBZ in food samples. The rabbit IgG based ELISA had a half-maximum inhibition concentration (IC50) of 7.41 ng mL-1, with a limit of detection of 0.27 ng mL-1. The ELISA showed negligible cross reactivity (<1%) with structural analogs, including hydroxy-MBZ, albendazole, oxfendazole, fenbendazole, flubendazole and oxfendazole, except the value of 90.5% for amino-MBZ. The average recoveries of MBZ spiked in chicken and mutton muscle samples by the assay ranged from 84.31% to 106.28% and agreed well with those of high-performance liquid chromatography. The above results indicated that the generated anti-MBZ IgG-based ELISA showed promise in routine screening analysis of MBZ residues in food samples.