Interleukin-8 (IL-8), a chemokine that participates in the early inflammatory process, plays a key role in mediating resistance to infection in animals. However, there has been no information about IL-8 at the protein level for characterizing the regulatory role of this molecule during the immune response in fish. In this work, we constructed a prokaryotic expression system and prepared a polyclonal antibody against IL-8 of ). The coding region without a signal peptide sequence was first amplified and cloned into pET-32a(+), a prokaryotic expression plasmid. The recombinant plasmid was then transformed into Escherichia coli Rosetta. A soluble fusion protein was expressed after induction by isopropyl β-D-1-thiogalactopyranoside. The purified protein was detected as a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with a predicted molecular mass of 27.8 kD, suggesting that the prokaryotic expression system for IL-8 of crucian carp was successfully constructed. The purified recombinant protein was used as an immunogen to raise polyclonal antibodies in New Zealand rabbits. The serum antibody titer reached 1:105, as detected by indirect enzyme-linked immunosorbent assay. The antibody was purified by affinity chromatography and had a high binding activity and specificity for the recombinant protein antigen, as measured by Western Blot. Immunohistochemistry results showed that the polyclonal antibody can also specifically bind to endogenous IL-8 in crucian carp tissues. Comparing the results of immunohistochemistry with that of fluorogenic quantitative polymerase chain reaction, the expression of IL-8 was consistent between the protein and mRNA levels, and its expression ranked among tissues as follows:muscle > spleen > head kidney > intestine. This study provides a material basis for further research on the role of IL-8 in the immune response of .