Objective：To develop the bacterial expression，purification and biological activity evaluation methods for Polo-like kinase 1 Polo-box domain（Plk1 PBD） protein. Methods：HeLa cDNA was served as a template to amplify Plk1 PBD gene fragment by PCR. After inserting into pET-28a vector，E.coli Rosetta（DE3） competent cells were transformed with pET-28a-Plk1 PBD plasmid. Plk1 PBD protein was expressed after induction and purified with HisTrap affinity chromatography，and then the biological activity was e－valuated by enzyme linked immunosorbent assay（ELISA） and fluorescence polarization（FP） assay. Results：The DNA sequencing and double digestion map for recombinant plasmid showed the construction was successful. SDS-PAGE and Western blot assay demon－strated that Plk1 PBD protein was successfully expressed and purified，and ELISA and FP assay showed a perfect biological activity of Plk1 PBD protein. Conclusion：Plk1 PBD protein is prepared successfully，which paves the way for further study on its cellular functions in vitro and the development for novel Plk1 inhibitors targeting Polo-box domain.