Objective To discuss the clinical application value of microplate allochroic silica gel assay for rapidly detecting rifampicin(RIF)-resistance of Mycobacterium tuberculosis(MTB).Methods Fifty MTB clinical isolates preserved in the tuberculosis(TB) laboratory of Nantong Municipal Sixth People's Hospital were detected RIF-resistance by using the microplate allochroic silica gel assay and compared with the Bactec MGIT960 method.Then,the RIF susceptibility test in 40 clinical sputum smear-positive specimens were simultaneously detected by using the microplate allochroic silica gel assay and Bactec MGIT960.Finally the sensitivity,specificity and accuracy of the test results were compared.Results The optimal inoculation volume of MTB was 10-3 mg/mL,the optimal detection time was 7-10 d and the judging critical value of the RIF minimum inhibitory concentration(MIC) Was 1.00 μg/mL by microplate allochroic silica gel assay.With the Bactec MGIT960 test as the gold standard,the sensitivity,specificity and accuracies of microplate allochroic silica gel assay for RIF-resistance susceptibility test of smear-positive specimens were 94.12％,100％ and 97.37％ respectively.Conclusion Microplate Allochroic silica gel assay can be used for directly detecting the MTB sensitivity to RIF of in sputum specimens.