Japanese encephalitis virus (JEV) (family Flaviviridae, genus Flavivirus) is an arbovirus of public health importance. The envelope glycoprotein of JEV is associated with viral attachment and fusion with host cell, determine the virus′s hemagglutination ability, cellular tropism, viral virulence, and induction of protective immune response. The domain Ⅲ of envelope protein (E protein) is an important region in inducing neutralizing antibodies against JEV. In order to study the antigenic structure of domain Ⅲ on E protein, domain Ⅲ of the envelope protein was expressed by fusion with GST in a pGEX-6p-1 vector. Western blot demonstrated that expressed fusion protein could be recognized by anti-JEV sera. To map the antigenic epitope of this region, a set of 14 partially overlapping short peptides spanning the domain Ⅲ were designed and expressed in fusing with GST. Then Western blot and ELISA reactivity of these short peptide fusion proteins to anti-JEV sera were surveyed, respectively. Four linear antigenic epitopes, E39 (³⁰⁵TEKFSFAKNPVDTGHG³²⁰), E45-1 (³⁵⁵VTVNPFVATSSA³⁶⁶), E48-1 (³⁷⁷PFGDSYIV³⁸⁴) and E49 (³⁸⁵VGRGDKQINHHWHKAG⁴⁰⁰) were identified. Immunization of mice with epitope fusion proteins revealed that all four proteins could elicit short peptide specific antibody. And in vitro neutralization test verified that E39 was linear neutralizing epitope. This result provides important basis for further structural and functional study of domain Ⅲ of JEV envelope protein, and development of diagnostic techniques and vaccines.