Objective To construct fused gene Ag85B-Esat6-HspX of Mycobacterium tuberculosis and investigate its expression in eukaryotic cells in vitro. Methods The gene encoding Ag85B，ESAT6 and HspX protein was amplified by PCR from genome of Mycobacterium tuberculosis H37Rv strain，and was inserted into cloning vector pUC19-T. After the sequence was confirmed，the fused gene was subcloned to eukaryotic expression vector pcDNA3. 1( － ) . After identified by restriction enzymec digestion and verified by DNA sequencing again，the recombinant plasmid pcDNA-Ag85B-Esat6-HspX was transfected into 293T cells with MegaTran 1. 0. Western blot was used to detect the expression of goal protein. Result A 65 kDa protein was detected with specific antibodies. Conclusion The eukaryotic expression vector pcDNAAg85B-Esat6-HspX has been constructed successfully and the fusion protein could be expressed specifically in 293T cells.