Lentiviral vectors were powerful gene delivery tools for gene therapy. We developed a new lentiviral vector pBobi-MIL that constitutively expressed O6-methylguanine-DNAmethyltransferase (MGMT) and Luciferase, linked by the internal ribosomal entry site (IRES), to realize drug tolerance and real time monitoring in vivo. All results from RT-PCR, drug treating clones forming, immunofluorometric assay and chemiluminescence detection showed that cells infected by recombinant lentivirus L-MIL simultaneously expressed these two genes. This lays the foundation for the further research in gene therapy and can also help identify lentivirus titer.