Objective To express the fusion protein of hOct4 and cell penetrating peptides using prokaryotic expression systems, and to optimize its expression methods and observe the membrane penetrating ability of the fusion proteins. Methods The pET-based prokaryotic expression system was constructed by genetic engineering, and the fusion plasmid was transferred into E.coli BL21(DE3) and Rosetta2（DE3）. The protein was purified by Ni affinity chromatography and identified by Western blotting analysis. The penetrating ability of the Rhodamine-labelled fusion protein was investigated using BJ cells. Results We successfully constructed pET21a(+)-hOct4-11R-His and pET21a(+)-EGFP-11R-His vectors. Fusion proteins hOct4-11-His and EGFP-11R-His were generated by transfering the plasmids into E.coli. The fusion protein was verified by Western blotting analysis and was detected in BJ cells. Conclusion We have successfully generated EGFP-11R-His and hOct4-11R-His fusion proteins, and the proteins can effectively enter the BJ cells and locate around the nuclei.