Infections with Flavobacterium oreochromis pose risks to Colossoma macropomum production. Since no treatment exists, prophylaxis through vaccination is the only form for disease control. The enzyme-linked immunosorbent assay (ELISA) is widely used in aquaculture to assess animal immunity and mainly to validate vaccination protocols. The method based on the use of immunoglobulin Y (IgY) is an alternative to consider due to its advantages over antibodies produced by small mammals. This study aimed to standardize an indirect ELISA (iELISA) protocol to validate the immunization of C. macropomum against F. oreochromis. A hen and fish were immunized to obtain anti-C. macropomum and anti-F. oreochromis antibodies, which were used to standardize the test. The iELISA was composed of the inactivated bacteria in the coating step, C. macropomum anti-Flavobacterium oreochromis as the primary antibody, and anti-C. macropomum IgY as the secondary antibody. The results demonstrate that the highest antibody titers were among the immunized groups, which were bacterin + adjuvant and bacterin. The iELISA enabled the detection of antibodies produced by C. macropomum, which were mediated by IgY specificity.