An enzyme-linked immunosorbent assay(ELISA) was developed to determine the extent of β-lactoglobulin (β-Lg) denaturation in heat treated milk. Polyclonal antibody was produced from the rabbit immunized with the β-Lg as an immunogen and was purified by the protein A affinity column. The antibody titer was determined above 1.28 × 10^5 in indirect ELISA. From the standard curve of indirect competitive ELISA for β-Lg. the sensitivitiy was found 90ng/㎖. Coefficient variations of intra- & inter-assay were 4.5-12% and 5.7-9.5%, respectively. When the ELISA of β-Lg were plotted against dilution of whey protein and skim milk, dilution assay curves could be made up to 40,000 times dilution of sample. When standard β-Lg was heated at 100℃ for longer time(5min. 15min & 30min). antibody-antigen reaction was decreased and the extent of heat-denatured β-Lg was reflected the difference of interval in dilution assay curve. When the whey protein and skim milk were heated respectively for various time(5min. l5min & 30min) at 100℃ the patterns of dilution assay curve were obviously displaced as compared with those of the whey protein and skim milk unheated and according to heating times, also made difference between dilution assay curves. When the skim milk was heated at various temperatures (65℃, 75℃ & 85℃) for 30min, according to higher temperature the patterns of dilution assay curve were showed apparent discrepancy against those of unheated skim milk.