Abstract: Objective To construct the pcDNA3.1-VR-3 eukaryotic expression vector for VEGFR-3 extracellular domain gene. The expression and identification of the vector was carried out in vitro. Methods The extracellular domain of VEGFR-3 encoding sequence was amplified by reverse transcriptase-polymerase chain reaction from C57BL/6 mice embryo and cloned into the HindⅢ-Xba Ⅰ sites of pcDNA3.1. After confirmed by sequencing the recombinant plasmid pcDNA3.1-VR-3 was transfected into COS-7 cells and its protein expression was identified by Western blot. Results The extracellular domains of VEGFR-3 encoding sequence was successfully cloned from C57BL/6 mice embryo. And the pcDNA3.1-VR-3 eukaryotic expression vector was constructed, which can be expressed in COS--7. Conclusion We successfully constructed the pcDNA3.1-VR-3 eukaryotic expression vector which may pave a way for further studies in animals experiment.