The present application provides a nucleic acid detection reagent and detection method for Mycobacterium tuberculosis, comprising: Tris-HCL, MgCl2, UNG enzyme, Bst DNA polymerase, reverse transcriptase, trehalose, sucrose, mannitol, Tween 20, guanidine hydrochloride and the primers of six kinds of Mycobacterium tuberculosis, the primers of six kinds of Mycobacterium tuberculosis are TB-F3, TB-R3, TB-FIP, TB-BIP, TB-LF and TB-LB, wherein TB is tuberculosis branch bacilli. In the embodiment of the present application, the nucleic acid detection reagent can be used to perform a one-step detection method for the nucleic acid (DNA or RNA) of pathogens present in throat swabs, nasal swabs, saliva, etc., thereby realizing a convenient, efficient and rapid detection, to achieve the sample in, the result out.