Carbofuran is a highly toxic insecticide and has been banned or restricted for pest control in many countries. Being a potent acetylcholinesterase inhibitor, carbofuran and its metabolite 3hydroxycarbofuran (3-OH-CBF) are required for the analysis of their residues in food. However, an immunochemical method for simultaneous analysis of carbofuran and 3-OH-CBF is still not available. Herein, we report an enzyme-linked immunosorbent assay (ELISA) based on a broad-specific monoclonal antibody (mAb) for simultaneous detection of carbofuran and 3-OH-CBF. The mAb, designated as 2E3, against both carbofuran and 3-OH-CBF was used to develop an indirect competitive ELISA (icELISA) with 50% inhibition concentrations of 0.76 and 0.69 ng/mL, respectively. The developed icELISA was validated by UPLC-MS/MS and is suitable for the rapid and simultaneous detection of carbofuran and 3-OH-CBF in fruits and vegetables.