Objective To compare the phage based splitting assay and BACTEC 460 in the rapid detection of Mycobacterium tuberculosis ( M. tuberculosis ). Methods 30 clinical isolates of M. tuberculosis, 10 strains of non M. tuberculosis, 7 strains of non mycobacterium and 60 sputum specimens of pulmonary tuberculosis patients were detected with phage based splitting assay and BACTEC 460 system. Results All the strains of M. tuberculosis clinical isolates detected with phage based splitting assay were positive, while all of the non M. tuberculosis and non mycobacterium strains were negative. 41 sputum specimens with BACTEC 460 culture positive and 19 sputum specimens with BACTEC 460 negative were detected with phage based splitting assay, the number of positive specimens was 34 (82.9%) and 5 (26.3%) respectively.Conclusions The phage based splitting assay can detect the M. tuberculosis easily and quickly in two days with high sensitivity and specificity.