Abstract The main cellular responses of innate immunity are phagocytic activity and the respiratory burst, which produces a high amount of reactive oxygen species. Natural killer enhancing factor (NKEF) belongs to the peroxiredoxin family that has an antioxidant function and enhances cytotoxic cell activity. This molecule may play a key role in macrophage and cytotoxic cell communication during the innate immune response of fish against pathogens. In fish, the NKEF gene has been characterized in some species as showing an up‐regulation in infected fish, suggesting a trigger effect upon NK‐like cells. To detect and localize this molecule in salmonids at protein level, a monospecific polyclonal antibody was generated. A probable NKEF‐like protein epitope region was identified and characterized using bioinformatic tools, and the sequence was chemically synthesized using Fmoc strategy, analysed by RP‐HPLC and its molecular weight confirmed by mass spectrometry. The synthetic peptide was immunized and antibodies from ascitic fluid were obtained. The resulting antibody is a versatile tool for detecting NKEF by different immune techniques such as ELISA, Western blotting and immunohistochemistry. Analysis of NKEF‐like protein is a useful method for characterizing immune properties of this molecule in fish during response to pathogens.