Edwardsiella tarda is a fish pathogen that causes systemic infections in fresh water and marine fish. Determining the antigenic proteins is important for the development of an immunodiagnostic tests and a vaccine for effective infection control in fish. In the current study, antigens were detected by immunoblotting and affinity column chromatography using a Japanese flounder (Paralichthys olivaceus) antibody produced by experimental infection with E. tarda. GroEL, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), outer membrane protein A, filament protein, 30S ribosomal protein S6, 50S ribosomal protein L9, cold shock protein, and carbon storage protein were identified as antigens of E. tarda through biochemical analyses of the molecular weights, isoelectric points, and N-terminal amino-acid sequences. These proteins can be easily detected in flounder infected with E. tarda and are potential diagnostic markers.