Listeriolysin O (LLO) is a heat-labile hemolysin produced by Listeria mono-cytogenes. Its hemolytic activity has been evaluated qualitatively by sodium dodecyl sulfate (SDS) electrophoresis and immunoblotting. In this experiment, an enzyme-linked immunosorbent assay (ELISA) was developed for quantitative analysis of LLO by using Streptolysin O (SLO) and antistreptolysin O (ASO) as the reagents. The selected coating and blocking buffers were 0.05 M Tris buffer (pH 8.5) and 0.25% casein solution with phosphate-buffered saline solution + 0.05% Tween 20 (PBS-T), respectively. A relationship between ASO and antibody was achieved with 5 mg/ml ASO and a 1:1,000 dilution of conjugate. The heat stability of LLO at 48, 62, 72, and 80C was examined by using this method and compared with a traditional hemolysis assay. Although the LLO is inactivated easily at those temperatures, the protein structure was not affected at temperatures lower than 80C for 3 min, pointing to a need for both hemolysis and ELISA to be conducted in determining both the activity and presence of LLO in foods.