Total RNA of leukocytes from orange-spotted grouper, Epinephelus coioides were extracted by Tripure kit. The first strand cDNA was synthesized by using MMLV reverse transcriptase. Then the cDNA were amplif ied by LD- PCR with SMART ó Oligonucleotide and CDS ó as Primers. After proteinase K digestion , Sfi . digestion, chromatography through CHROMASPIN- 400, over 500bp fragments were collected and ligated with KTripIEx2 vector. After package in vitro, cDNA library containing 1. 65 @ 106 private clones was constructed, in which more than 82% clones were recombinant . After amplif ication, the titer of the library was 7. 5 @ 109 pfu#mL- 1. The insert DNA were between 500 and 2300bp, and most of them were between 750 and 1000bp. The library can be used to screen cytokine genes of orange-spotted grouper.