Objective: The recombinant eukaryotic vector for gene encoding outer membrane protein P6(P6) of the nontypeable Haemophilus influenzae (NTHi) was constructed and expressed in HeLa cells for the sake of providing a foundation to develop the nucleic acid vaccine. Methods: The target gene fragment encoding the P6 was amplified from the genome of the NTHi which was used as the template, then the plasmid pcDNA3.1/HisA and the P6 fragment was digested with endonucleases simultaneously, purified and connected. The recombinant vector pcDNA3.1/His A-P6 was used to select, transform and express in HeLa cells. Meanwhile, the expression of this eukaryotic vector in HeLa cells was investigated by using fluorescence microscopy. Results: As demonstrated by PCR, restriction endonuclease analysis and sequencing, the size of the inserted target gene fragment was found to be P6. And this gene fragment was proved to be the gene encoding the P6 protein in HeLa cells by means of fluorescence microscopy. Conclusion: It is evident that the recombinant eukaryotic vector pcDNA3.1/HisA-P6 has been constructed and successively expressed in HeLa cells.