Background: Tumour necrosis factor-alpha (TNF-α) is a pro-inflammatory cytokine which exerts a variety of immunological functions in vertebrates. TNF-α has been identified and cloned in a number of teleost fish species; nevertheless, the functions displayed by this cytokine in fishes remain poorly understood, given that the low sequence identity compared to their mammalian counterpart, limit fish TNF-α detection using mammalian antibodies. Then, for fish immune response characterization is fundamental the production of specific fish anti-TNF-α antibody. Results: We have developed a monoespecific antibody against the pro-inflammatory molecule TNF-α of salmonid fish. TNF-α epitope region was identified and characterized using bioinformatic tools. The epitope sequence was chemically synthesized using Fmoc strategy, analyzed by RP-HPLC and its molecular weight confirmed by mass spectrometry. The synthetic peptide was used to immunize mice and antibodies from ascitic fluid were purified. The resulting antibody was used for molecular and histochemical detection in gut samples from salmonid fishes treated with different food. By ELISA, we detected a differential expression of TNF-α, the western blot analysis shows recognition of the whole TNF molecule and by immunohistochemistry TNF-α positive cells were observed. Conclusions: We provide an immunological tool, validated through classical immunological assays, which can be a useful tool for characterizing fish TNF-α function.