In this paper, we report the development of a sensitiveand specific monoclonal antibody-based immunodiagnosticmethod for the detection of iso-tenuazonic acid (ITeA),an Alternaria mycotoxin, in food samples. The ITeA wasderivatized with hydrazine hydrate to produce the antigen(E)-3-(1-hydrazonoethyl)-4-hydroxy-5-isobutyl-1H-pyrrol-2(5H)-one (ITeAH) which was further reacted with glyoxalicacid to generate the hapten (E)-2-((Z)-(1-(4-hydroxy-5-isobutyl-2-oxo-2,5-dihydro-1H-pyrrol-3-yl)ethylidene)(ITeAHGA) which was used as an immunogen after conjugationto bovine serum albumin (BSA). A highly specific monoclonalantibody selectively binding to ITeAH was generatedvia the hybridoma technique and subsequently used to constructa heterologous indirect competitive enzyme-linked immunosorbentassay (icELISA) using ITeAH as the competitiveantigen for the detection of ITeA with a limit of detection(LOD) of 0.5 ng/mL. Under the optimum conditions, the developedicELISA is highly sensitive (IC50 = 7.8 ng/mL) withrecovery rates ranged from 82.3 to 109.8% for spiked foodsamples. The comparative analysis of results revealed a goodcorrelation between the icELISA and the standard HPLC-MS/MS method, confirming the suitability of the developedicELISA for screening and detection of mycotoxin ITeA infood samples.