Objective:To establish a method of rapidly detecting Mycobacterium tuberculosis resistance to streptomycin and ethamtubol by gene-chip. Methods:To detect the SM and EMB resistance of 160 clinical isolates of M.tuberculosis by Gene Chip, and compare them with the results of the BACTEC960 method. Then validate the concordance and discrepancy result of the two methods by DNA Sequencing method. Results:Of 160 strains of M. tuberculosis clinical isolates, 111 strains were SM-susceptible and 49 strains were SM-resistible with Gene Chip while 100 strains were SM-susceptible and 60 strains were SM-resistible with BACTEC 960 method; 142 strains were EMB-susceptible and 18 strains were EMB-resistible with Gene Chip while 130 strains were SM-susceptible and 30 strains were EMB-resistible with BACTEC 960 method. According to the result amended by the BACTEC 960 method, the sensitivity, specialty and veracity were 80.0%, 99.0%, 91.9% and 60.0%, 100% and 92.5% respectively. The most frequent mutant sites are rpsL 43, 88 aag→agg in relation to SM-resistible and embB 306 atg→gtg in relation to EMB-resistible. Conclusion:Gene Chip method could detect the SM and EMB resistance of MTB easily and rapidly. At the same time it showed higher specialty and sensitivity, and could be used for clinical detection of SM-resistant and EMB-resistant strains.