To study biological activities of Duck Interferon Alpha (DuIFN-α) and prepare antivirus medicine, the eukaryotic expression vector of mature polypeptide of Duck Interferon Alpha (mDuIFN-α) gene was constructed and expressed in insect cell. By means of PCR technique, the mDuIFN-α gene was cloned from pMD-18-duIFN-αrecombinant. The gene was then inserted to pGEM-T vector and identified by restriction endonuclease analysis and sequencing. The mDuIFN-α gene was ligated with the eukaryotic expression vector pMelBacA, then transfected into Sf9cell line. Recombinant polypeptide was effectively expressed in insect cell and its molecular weight was 34 ku.