Abstract: This study aimed to prepare and characterize the monoclonal antibodies (McAbs) against the membrane proteins in theronts of Cryptocaryon irritans. For the study, Balb/c mice were immunized with the proteins extracted from the theronts. Spleen cells from the mice were fused with SP2/O cells to confirm the presence of hybridoma cells using indirect ELISA. Then, the ascites containing McAbs were collected from the mice containing hybridoma cells for antigen identification in Western-blot and immunofluorescence assays. Three hybridoma antibodies, i.e., 5D11AG5, 5H9BG3 and 6E11CE7, were found to consistently induce the target antigens. These McAbs in the mouse ascitic fluids were all IgM type. The titers of the 3 McAbs ascites were 1:3200, 1:25600 and 1:51200, respectively. Western-blot assay indicated that McAb-5D11AG5 and McAb-5H9BG3, in particular, recognized the proteins with a molecular weight approximating 35 kDa. The peptides recognized by McAb-5D11AG5 had a high homology with the surface antigens of C. irritans and the tubulin of Tetrahymena thermophila. According to the immunofluorescence assay, McAb-5D11AG5 and McAb-5H9BG3 recognized the antigens mostly on the front, while McAb-6E11CE7 on the external surface, of the membrane. The results obtained would aid further study to separate and screen the functional proteins of C. irritans.