Abstract: Objective To construct eukaryotic expressing recombinant vector of canine transferrin receptor gene (TfR ), then to transfect Chinese hamster ovary (CHO) cells with the vector for establishment of stable expression of TfR in CHO cell line. Methods The full-length TfR fragment was amplified by RT-PCR from the canine cells (walter reed dog cell, WRD) and then inserted into eukaryotic expression vector pCDNA3. After identification with enzyme digestion and sequencing, the recombinant vector was transfected into CHO cells by TransLipid Transfection Reagent. The stable transfected CHO cell line was then established by screening cultures with G418, and the expression of TfR was identified by RT-PCR, Western blot and immunofluorescence, respectively. Results The eukaryotic expression vector pCDNA3-TfR was constructed successfully by checking with enzyme digestion and sequencing, and the highly expressed canine TfR was observed in CHO cells transfected with pCDNA3-TfR by using RT-PCR, Western blot and immunofluorescence, respectively. The stable CHO cell line with canine TfR expression was established. Conclusion The construction of the eukaryotic expression vector pCDNA3-TfR and the establishment of stable CHO cell line with TfR expression provide solid foundation for further experimental studies on the function of TfR.