In this study we developed a rapid and simple assay to detect RFP in fish using an immunochromatographic assay. We produced a sensitive and selective monoclonal antibody against RFP with a half-maximal inhibitory concentration (IC50) of 10.7 ng/mL. Fish samples were extracted with ethyl acetate, then centrifuged at 8000 rpm for 10 min, and concentrated by drying over nitrogen. We found under optimal conditions, that the RFP cut-off limits for the test strip was 10 µg/mL in both 0.01 M phosphate buffered saline and fish samples, and each test could be completed within 5 min. Therefore, immunochromatographic test can be used as a reliable, rapid, and sensitive method for the detection of RFP in fish.