A biotin-labeled antigen (BLA) was adapted to a sandwich enzyme-linked immunosorbent assay (S-ELISA) for detection of Japanese encephalitis (JE) antibody in a variety of animal sera. JE antigen was fixed on the wells of a microplate and became bound to the specific antibody which could react with a peroxidase-labeled avidin conjugate and azino-di-(3-ethylbenzthiazolin sulfonic acid) (ABTS) as a substrate. The BLA-S-ELISA could simultaneously detect JE antibody in all hemagglutination inhibition (HI) positive sera from man, swine, monkey, horse, cattle, rabbit, rat, mouse and pigeon by using the same reagents under the same test conditions. The antibody titers obtained by BLA-S-ELISA in human and swine sera corresponded well with HI antibody titers. The sensitivity of BLA-S-ELISA appeared to be higher for IgM antibody than for IgG antibody. Since the non-specific reaction was extremely low in BLA-S-ELISA, the cut-off titer for the assay could be set as low as 1:2.5 of serum dilution for positive antibody.