We improved the dot enzyme-linked immunosorbent assay (dot-ELISA) reported by Itoh and Sato, and assessed the usefulness of this test for the diagnosis of amebiasis. The sensitivity of dot-ELISA was compared with that of plate ELISA, the indirect hemagglutination test (IHA), and the indirect fluorescent antibody test (IFA) for the diagnosis of amebiasis. Of 37 serum samples from patients with documented amebiasis, 36 (97.3%) were positive by dot-ELISA. There was consistency among the results of dot-ELISA, plate ELISA, and IFA, although the positive rate of IHA was lower than that of the others (78.4%; 29 of 37 cases were positive). The specificities of dot-ELISA and plate ELISA were assessed using a total of 68 sera, collected from 38 patients infected with seven different parasites other than Entamoeba histolytica, 10 patients showing diarrhea or liver abscess without parasitic infection, and 20 healthy individuals. The two assays showed no false-positive results. There were no differences in sensitivity and specificity between dot-ELISA and plate ELISA. However, the dot-ELISA technique seems to be more feasible for clinical application than plate ELISA techniques, because the assay does not require any specific equipment.