With the persisting increase of drug-resistant stains of M. Tuberculosis around the world, rapid and sensitive detection of antibiotic of M. Tuberculosis is becoming more and more important. In the present study, drug susceptibility of M. tuberculosis were detected by recombinant mycobacteriophage combined with bioluminescence sensor. It is based on the use of recombinant mycobacteriophage which can express firefly luciferase when it infects viable mycobacteria, and can effectively produce quantifiable photon. Meanwhile, in mycobacterium. cells treated with active antibiotic, no light is observed. The emitted light is recorded by a bioluminescence sensor, so the result of drug-resistant test can be determined by the naked eye. 159 stains of M. Tuberculosis were applied to this test on their resistant to rifampin, streptomycin and isoni*azid. It is found that the agreement of this assay with Lowenstein-Jensen slant is: rifampin 95.60%, isoniazid 91.82%, streptomycin 88.68%, which showed that it is a fast and practical method to scan and detect drug resistant of mycobacterium stains.