A rapid and accurate diagnosis increases the treatment effect and decreases the mortality of tuberculosis (TB) patients. The purpose of this study was to establish an accurate, unique, and rapid molecular diagnostic technique to screen Mycobacterium tuberculosis (MTB) from clinical sputum. A unique gene in MTB strains called conserved protein TB18.5 (TB18.5) was selected by bioinformatics analysis. Two pairs of primers were designed to amplify TB18.5 using the nested polymerase chain reaction (PCR) or quantitative real-time PCR. Nine pathogens and the MTB strain were used to determine the specificity of the TB18.5 gene. The sensitivity assay was performed after optimizing the PCR conditions. The correct fragment was amplified when a 10 copy number template was used. A total of 232 sputum samples were collected from TB patients (from 2019 to 2020) to evaluate the accuracy of the molecular method in this study. MTB was first detected using the BACTEC MGIT-960 culture test and the Gene Xpert MTB/RIF assay. Totals of 195 (84.05%), 182 (78.45%), and 162 (69.83%) sputum samples were determined to be infected with MTB using nested PCR, the Gene Xpert MTB/RIF assay, and the BACTEC MGIT-960 culture test, respectively. In summary, a rapid, unique, and sensitive molecular method was established to diagnose TB infection in clinical sputum samples.© 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.