In order to rapidly diagnose and differentiate tuberculosis from other bacterial infections, a 16S rRNA gene (16s rDNA)-directed multiplex PCR system was developed. In this system, a pair of universal primers and a tubercle bacillus (Tb)-specific primer were designed based on highly conserved regions and Tb species-specific variable region of bacterial 16s rDNA. A 360bp fragment was detected in all bacteria tested, and a 210bp fragment was found only in Tb. 19 species of known bacteria including Tb were used for evaluating specificity, universality and sensitivity of the PCR. Candida albicans and human diploid cell served as controls. It was found that both 210bp and 360bp fragments were amplified only in Tb, and only 360 bp fragment was detected in other 18 species of general bacteria. Candida albicans and human cells were negative for both 360bp and 210bp fragments.The lowest detectable level of the PCR was 10 fg of DNA for Escherichia coli and 100 fg of DNA for Tb. The results indicated that this multiplex PCR system for the simultaneous detection of Tb and other common bacteria had higher specificity and sensitivity, as well as good universality and might be useful to rapidly diagnose bacterial infections and effectively distinguish tuberculosis from other bacterial involvement.