The invention discloses a kit of Mycobacterium tuberculosis and a detection method thereof. The kit contains an enzyme-linked immunosorbent assay (ELISA) plate, a biotin-labeled single-stranded DNA adaptor, horseradish peroxidase-labeled streptavidin and a chromogenic substrate. In the detection method provided by the invention, the biotin-labeled single-stranded DNA adaptor of Mycobacterium tuberculosis H37Rv is used as a probe and a method which is similar to the indirect enzyme-linked immunosorbent assay (ELISA) method is adopted to detect Mycobacterium tuberculosis; and Mycobacterium tuberculosis or sputum to be detected or a body fluid sample, and a control sample are added in the ELISA plate for incubation, Mycobacterium tuberculosis combined with the plate is trapped by the biotin-labeled single-stranded DNA adaptor and then washed, the horseradish peroxidase-labeled streptavidin is added, the substrate is added for developing, and the optical density (OD) value is detected by a microplate reader after developing when the absorption wavelengh is 450nm. When the kit provided by the invention detects Mycobacterium tuberculosis or the Mycobacterium tuberculosis or somatic antigen in sputum, blood and body fluid, the detection speed is high, the detection is convenient and safe, the detection time can be saved, the efficiency is high, the cost is low, the detection sensitivity and accuracy are high, and the application prospect is wide.