The present invention provides a kit for rapid detection of Mycobacterium tuberculosis in a sputum sample, and the kit includes a liquefied solution, a lysis solution, a probe solution, a stop solution, a fixative solution, a carrying glass sheet and a cover glass sheet. After liquefaction of the sputum sample, cell lysis, probe hybridization and fluorescence fixing and other operations are performed, and a fluorescence microscopy is used for macroscopic observation, counting and analysis of the Mycobacterium tuberculosis. The kit is simple in operation method, short in procedure, easy to operate, strong in test result specificity, high in sensitivity, clear in results and high in reliability, and can be used for direct, simple and effective detection of the Mycobacterium tuberculosis in the sputum sample.