The invention relates to a joint detection method for the drug resistance of mycobacterium tuberculosis and the pyrazinamide thereof in a clinical sample, comprising the following steps of: A, extracting DNA (deoxyribonucleic acid) in the sample; B, detecting the mycobacterium tuberculosis via fluorescent quantitative PCR (polymerase chain reaction) amplification for a tuberculosis genome segment containing total-length pyrazinamide enzyme genes (pncA); and C, detecting the drug resistance of pyrazinamide for the mycobacterium tuberculosis positive sample, wherein the steps for detecting the drug resistance of the pyrazinamide are as follows: 1, performing PCR reaction once by taking the fluorescent quantitative PCR product as a template, so as to add an in-vitro expression element; 2, adding the PCR product in a cell-free expression system, so as to express a pyrazinamide enzyme; and 3, comparing the pyrazinamide enzyme activity of the sample with a standard strain, so as to give the drug resistance result of pyrazinamide. According to the invention, joint detection for the drug resistance of mycobacterium tuberculosis and the pyrazinamide thereof in the sample is realized by combining fluorescent quantitative PCR amplification for pncA genes with the enzyme activity of the in-vitro expressed pyrazinamide enzyme, without the need of bacterial culture and a DNA sequencer.