A drug-resistance gene film chip for detecting mycobacterium tuberculosis is prepared by designing 54 probe spotting on a nylon film by aiming at the mutant sites of mycobacterium tuberculosis rpoB, kagG, embB, inhA, ahpC, gyrA, rrs, rpsL and pncA gene; 12 pairs of specific primers with biotin-labeled at 5' end are utilized; a sample DNA is subjected to triple PCR and amplified to form a large amount of gene fragment products with biotin; the amplified products and the probes on the film chip carry out specific hybridization; and then film washing, enzyme-linking and color reaction are carried out, thus preparing the chip. The gene film chip and the detection method thereof can detect the common gene mutations of the mycobacterium tuberculosis on the drug resistance of drugs such as isoniazid, rifampicin, streptomycin, ethambutol, pyrazinamide, quinolone and the like at one step, and are applicable to extracorporeal detection sputum sample, clinical isolation strains and mycobacterium tuberculosis multi-drug resistant gene in the organization sample.