Background: Different immunological methods are used for the serodiagnosis of Fasciola hepatica infections, however, they have limited specificity and/or sensitivity.Objective: To improve the specificity of ELISA using a partially modified method for preparation of excretory-secretory (ES) antigens.Methods: Studied groups were as follows: 75 patients with parasitological evidence of fascioliasis; 250 samples from normal individuals from non-endemic areas with no history of fascioliasis; 50 patients who were infected with other parasites. ES was prepared by incubation of adult worms in protease inhibitor containing media. ELISA was performed using this antigen. The antigenic components of ES were revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis.Results: The sensitivity and specificity of the test were 100% and 99.7%, respectively. Conclusion: Our results showed that the ES of adult worms of Fasciola hepatica is a suitable antigen for ELISA procedure.