Abstract: Total RNA was extracted from small intestine of New Yangzhou laying hens at 200 day of age. RT-PCR was conducted subsequently based on published chicken Calbindin-D28k gene sequence (NM_205513.1). The expected amplified cDNA fragment was cloned into TA vector. Sequence analysis revealed that the cloned Calbindin-D28k gene, encoding 262 amino acids, was 789 bp in length which was 99.9% identity but only one nucleotide changed from G to T at site 760 bp compared with published sequence from GenBank database. The homology of Calbindin-D28k gene sequence between chicken and other species such as horse，human，mouse and frog was 79.3%、79.1%、77.4%、77.3%, respectively. Reconstruction plasmid of pCEP4-CaBP-D28k was carried out by inserting the fragment of CalbindinD28k gene into eukaryotic expression vector pCEP4, and the target segment in recombinant plasmid was characterized and confirmed by restrictive endonuclease assay as well as sequence analysis. This research provide a novel means to study regulation mechanism of calcium metabolism in chickens through pCEP4-CaBP-D28k.