In order to explore the regulation mechanism of flowering, the flowering gene CONSTANS gene, designated as PyCO (GenBank accession number: KF246752), was isolated from Pyrus pyrifolia Nakai by homologous cloning strategy. Sequence analysis revealed that the full-length cDNA sequence of PyCO is 1023 bp and encodes 340 amino acids, molecular weight and isoelectric point of the protein is 37.81 and 5.95 respectively. The sequence of PyCO that contains B-box and CCT domain has the typical structure characteristics of CO gene in plants. According to the phylogenetic analysis of CO genes, deduced amino acid of PyCO gene demonstrates high homology with MdCO (93%), and more than 70% homologous with that in Prunus persica, Theobroma cacao and Fragaria vesca. In the end, the fusion protein was produced at a high level by prokaryotic expression and then 58.5 kD protein was obtained. This study will provide a foundation for further discuss the flowering mechanism of pear.