Abstract: According to the reports that polo-like kinase 3 can promote the growth, proliferation and migration of prostate cancer cells, this study was aimed at constructing the recombinant mammalian cell expression plasmid of pCDNA3-FLAG-PLK3, identifing the expression and localization of this recombinant protein in prostate cancer cells. The primers were designed and synthesized according to the full-length coding sequence of Polo-like kinase 3（PLK3）. Plasmid containing coding sequence of PLK3 was used as a template in PCR amplification, and the PCR product was digested by endonucleases EcoR Ⅰ and Xho Ⅰ. After ligation and transformation, monoclonal colony was picked and amplificated. Then, plasmid extraction was carried out, and the plasmid was confirmed by enzyme digestion and sequencing. Further, the recombinant plasmid with the correct sequence was transfected into CWR22 RV1 and LNCaP cells, and the expression of FLAG-PLK3 in CWR22 RV1 and LNCaP cells was detected by Western Blot experiments. Then immunofluorescence staining was performed to detect the localization of the exogenous FLAG-PLK3 in cells. In addition, Western Blot experiments was used to determine endogenous PLK3 protein expression in prostate cancer cell lines. The results showed that pCDNA3-FLAG-PLK3 plasmid was correctly constructed and expressed in prostate cancer cells. Exogenous FLAG-PLK3 was mainly distributed in the cell membrane, and also with a small amount in the cytoplasm. Endogenous expression of PLK3 protein was higher in DU145 cells, and lower in CWR22 Rv1 cells, than that of the other cell lines. This study provides an experimental basis for the further studies on the function and mechanism of PLK3 protein in prostate cancer cells.