A wide-host-range plasmid pTRUS containing a homologous rus gene under the control of Ptac promoter was constructed and transferred into Acidithiobacillus ferrooxidans ATCC19859 using conjugation gene transfer method to generate the engineered strain of A. ferrooxidans(pTRUS). The plasmid-based recombinant rus gene was successfully expressed. According to the results of real-time quantitative PCR assay, not only the transcription level of recombinant rus gene but also the transcription levels of the other genes of rus operon (cyc1, orf, coxB, coxA) were increased in A. ferrooxidans(pTRUS). The ferrous ion (Fe2+) oxidation activity was increased in A. ferrooxidans(pTRUS).