A hen's egg is one of the most common causes of food allergy. The allergen quantitation in hot-processed food is always a difficult task, because the protein in these samples will be denatured, insoluble, and degraded. This article presents a competitive enzyme linked immunosorbent assay (ELISA) for the quantitation of ovalbumin in hot-processed food. Its recovery was improved nearly two times by the assay method as compared with previous sandwich ELISA. The heat and DL-Dithiothreitol treated ovalbumin was used as antigen for monoclonal antibody preparation. A smaller labeled antibody molecule, horseradish peroxidase (HRP) labeled Fab' fragment, was used to replace IgG in ELISA for improving sensitivity and analytical speed of the method. A binding site protection procedure was developed for Fab' fragment labeling with HRP, which prevented damage to the Fab' binding site. The combination and separation steps were efficiently completed in an affinity spin column. Based on the optimized ELISA condition, the IC50 was 1.2 µg/mL and the coefficients of variation were less than 10%.