Objective  To construct the eukaryotic expression vector plasmid enhanced green fluorescent protein(pEGFP)-N1-CPNE3, and identify the expression and localization of Copine-3 protein in cells.    Methods  The Copine-3 coding sequences (CPNE3) was amplified by RT-PCR from human bronchial epithelial (HBE) cells and inserted into eukaryotic expression vector pEGFP-N1. The recombinant plasmid pEGFP-N1-CPNE3 was confirmed by endonuclease digestion and sequencing before it was transfected into 293T and H1299 cells. Cellular localization of Copine-3-EGFP fusion protein was detected by con-focal laser scanning. Expression of Copine-3 in 293T and H1299 cells was detected by Western blotting analysis. Localization of Copine-3 in clinical samples of the lung adenocarcinoma patients was detected by immunohistochemistry.    Results  CPNE3 was successfully constructed into the eukaryotic expression vector pEGFP-N1 and expressed in 293T and H1299 cells. Furthermore, the location of Copine-3 protein in cytoplasm and nucleus was determined by immunofluorescence staining, immuno Western blotting and immunohistochemistry in those cells and clinical samples.    Conclusion  The eukaryotic expression vector pEGFP-N1-CPNE3 is constructed successfully, and Copine-3 protein is localized in cytoplasm and nucleus.