The invention belongs to the field of molecular biology detection, relates to a mycobacterium tuberculosis rifampicin drug resistance detection method, and provides a method for detecting mycobacterium tuberculosis rpoB mutant genes. The method comprises the following steps of culture, extraction, amplification, purification, digestion, separation, gathering, image displaying and identification. Compared with the existing similar detection methods, the method of the invention adopts once culture, novel forward and backward primers are adopted, the purification step is adopted, the cracking digestion is adopted, the gathering step is adopted before the image displaying, a labeled digoxin antibody developing system is adopted, conventional equipment is adopted, and medicament can be easily obtained, so the method provided by the invention has the obvious advantages that the detection is fast, the detection rate is high, the map identification performance is good, the identification is accurate, the cost is low, and the like. The clinic diagnosis and treatment requirements can be more exactly and efficiently met.