Seropositivity against Japanese encephalitis virus (JEV) was measured by hemagglutination inhibition test (HI) on sera from 323 horses raised in Jeju. Enzyme-linked immunosorbent assay (ELISA) method was applied for the confirmation of its availability as a tool of detecting anti-JEV antibodies in horses. The positive rate scored 50.4%. The positive rates were increased according to ages. The highest peak was shown at October. And Jeju Ponies showed higher positivity compared to thoses of Thoroughbred horses. When compared the results obtained by HI and ELISA, there was slight correlations between the two methods (r=0.7100). Besides, ELISA could discriminate between true and false positive sera by neutralizing serum specimen with JEV antigen.