AIM To construct eukaryotic expression vector of human CD34 and transfect it to 3T3 cells so as to establish stably transfected 3T3 cell line. METHODS The RNA was extracted from KG1a. CD34 gene was amplified by RT-PCR. With the double-enzyme digestion, CD34 gene was cloned into pCI-neo eukaryotic expression vector, yielding pCI-CD34. The pCI-CD34 was transfected into 3T3 cell by electroporator. Stably transfected 3T3 cell line was established, and the CD34 expression in the transfected cells was detected by RT-PCR and FACS. RESULTS The eukaryotic expression vector pCI-CD34 was constructed, and stably transfected 3T3 cell line was established. CONCLUSION Construction of eukaryotic expression vector of CD34 and the establishment of stably transfected 3T3 cell line are helpful to preparation of anti-CD34 mAbs and further functional study of CD34.