Great blue-spotted mudskippers (Boleophthalmus pectinirostris) have lived in mudflats along the coasts of Japan and South China for a long time. Compared with the fish living in water, B. pectinirostris, as an amphibious fish, is more vulnerable to terrestrial and aquatic pathogenic factors. Interleukin-8 is a CXC chemokine that plays an important role in regulating the inflammatory response. In this study, we screened the interleukin-8 gene from skin transcriptome of B. pectinirostris, then cloned and verified its coding region. The results indicated that the open reading frame(ORF) of B. pectinirostris IL-8 gene was 306 bp, coding 101 amino acids, including a typical signal peptide of 18 amino acids, and a SCY domain of 62 amino acids, which also possessed the four conserved cysteine residues, namely Cys-30, Cys-32, Cys-57 and Cys-73. Like most fish, B. pectinirostris interleukin-8 amino acid sequence lacked the ELR motif which was the receptor binding site and it was replaced by Asn-Ser-His (NSH) in great blue-spotted mudskipper. In all known fish amino acid sequences, rules of this motif were as follows: the first amino acid was never the amino acid with positive charge, the second amino acid was generally those amino acids with no charge, and most of third amino acid were amino acids with positive charge. Phylogenetic analysis showed that purification selection affected the diversity of this motif in fish. Tissue expression analysis by RT-qPCR indicated that interleukin-8 gene was widely expressed in healthy tissues of B. pectinirostris and it had the highest expression in the gill and brain. Bacteria and poly (I∶C) injection experiments showed that expression of IL-8 in liver, spleen and brain tissues was up-regulated after the infection, indicating that IL-8 plays an important role in the inflammatory response and immune response of liver, spleen and brain tissues. This study provided important reference for the research of immune genes of B. pectinirostris, thus having more comprehensive understanding of the evolution process and function of fish immune genes.