Abstract: With the layers of mucus and nonspecific immune defenses, the fish gill is exposed to the external environment and forms the initial barrier to invasio n by pathogens. As one of the mucosa-associated lymphoid tissues in teleost fishes, gill contains populations of leucocytes. In order to probe the immune-related genes in red seabream (Pagrus major), the cDNA library of gill s was constructed from the red seabream inoculated with several species of pathogenic bacteria. The total RNA was extracted by TRIZOL and mRNA was isolated by biotin-oligo (dT). Then, cDNA was synthesized from 5 μg mRNA. Through running gel electrophoresis of double-strand cDNA, the fragments of 500-4000 bp were harvested, concentrated, ligated with λZAP vector and packaged in vitro. The primary cDNA library contained 1.75 ×10⁵ recombinants. The titer of amplified library was tested up to 1×10⁹ pfu·mL^-1, and the insert size of random picked phagemid was between 500-2000 bp. PCR with specific primers of hepcidin was performed to cloning of the homologous genes from cDNA library and a fragment of hepcidin cDNA sequence was amplified. The identity was above 85% in comparison with the hepcidin cDNA isolated from the liver of also red seabream. Therefore, the construction of cDNA library provides the basis for screening immune-related genes from red seabream.