Abstract: This experiment was conducted to explore the potential regulatory sequences of duck CD8α gene and the mechanism of transcription regulation. The CD8α gene promoter was cloned by genome walking, the sequence of the promoter was analyzed by online software of bioinformatics, and then the promoters from DHVresistant and DHVsusceptible duck were subcloned into the luciferase expression vector pGL3Basic directly, and their luciferase activity was detected. The DNA sequence of 2 480 bp was amplified including 56 bp in exon1 and 2 424 bp of promoter region. There were TATAbox, GCbox, CAATbox and transcriptional start site in the sequence. 41 transcriptional factor binding sites including CdxA, Nkx2, GATA1, SRY, and so on, were detected. The luciferase reporter gene recombinant pGL3 CD8α including correct target gene was constructed and identified by restrictive endonuclease enzyme cutting and sequencing, the pGL3Gy and pGL3Gk plamids had strong luciferase activity. The result lay a foundation for exploring the promoter activity and transcriptional regulation mechanism of duck CD8α gene.