We had previously developed a nested polymerase chain reaction (PCR)-immunochromatography test (ICT) for identification of Mycobacterium tuberculosis (MTB) and differentiation of MTB from other members of M. tuberculosis complex (MTBC) from clinical sputum samples (Soo P.C. et al., Journal of Microbiological Methods. 2006, 66(3):440-8.). To further improve the detection flexibility, simplicity and efficiency, and reduce the cost, in this study, an alternative molecular diagnosis assay that utilizes gold nanoparticles derivatized with thiol modified oligonucleotides was developed. The gold nanoparticles probes, GP-1/GP-2 for IS6110 and GP-3/GP-4 for Rv3618, were designed to specifically hybridize with target DNAs of MTBC and MTB strains, respectively. Efficacy of the gold nanoparticle probes assay was evaluated by directly and simultaneously detecting not only MTBC but also MTB from 600 clinical sputum specimens. Results were compared with traditional culture and biochemical identification methods together with patients' clinical assessments. This assay showed a 96.6% sensitivity and 98.9% specificity towards detection of MTBC, and a 94.7% sensitivity and 99.6% specificity for detection of MTB. In conclusion, the gold nanoparticle probes assay is a simple, rapid, cost-effective and accurate detection system and shows great potential in clinical application of MTBC and MTB detection, especially in developing countries.