A monoclonal antibody-based competitive indirect enzyme-linked immunosorbent assay (ELISA) with improved sensitivity and specificity for the determination of furaltadone metabolite 5-methylamorpholino-3-amino-2-oxazolidone (AMOZ) was described. AMOZ was derivatized with 2-(3-formylphenoxy)acetic acid and coupled with bovine serum albumin to form a novel immunogen. BABL/c mice were immunized and monoclonal antibody specific to the nitrophenyl derivative of AMOZ (NP-AMOZ) was produced and characterized. Four other haptens with different heterology to the immunizing hapten were synthesized and coupled to ovalbumin as coating antigens to study the effect of heterologous coating on assay sensitivity. Under the optimized heterologous coating format, the competitive indirect ELISA showed very high sensitivity to NP-AMOZ, with an IC(50) of 0.14 μg/L and limit of detection of 0.01 μg/L. The assay showed high specificity toward NP-AMOZ, and negligible cross-reactivity with analogous compounds was observed. The average recoveries of AMOZ from spiked fish and shrimp samples were estimated to range from 81.0 to 104.0%, with coefficients of variation below 20%. Good correlation was obtained between the results of ELISA analysis and of standard liquid chromatography-tandem mass spectrometry analysis. These results indicated that the proposed ELISA is ideally suited as a monitoring method for AMOZ residues at trace level.