Abstract: Objective：To establish and evaluate the direct matrixassisted laser desorption ionization-time of flight mass spectrometry（MALDI-TOF MS) for the identification of mycobacteria from positive liquid media. Methods：Sputum samples from 1 466 patients hospitalized in Beijing Tongren Hospital affiliated to Capital Medical University during June 2014 and May 2016 were collected, and cultured by the Bactec^TMMGIT^TM960. A total of 105 patients with mycobacteriapositive liquid media were further confirmed with ZiehlNeelsen acidfast staining and selected in our study. They were identified by the improved MALDI-TOF MS method（"Tongren") in which the culture fluid was performed pretreatment and then identified by MALDI-TOF MS directly and the classic method in which the culture fluid was transferred to the L-J solid media and the obtained colonies were identified by MALDI-TOF MS, respectively. The identification results were further verifed by 16S rRNA and 16S~23S rRNA intergenic region sequencing. Results：When the gene sequencing identification was used as the gold standard, the accordance rate between the direct and classic MALDITOF MS methods was 100%. The accuracy rates of the two methods at the genus level were 100%（105/105), and 99.05%（104/105) at the species level. There was no significant diffenrence in the proportion of identification score above 2.000 between the two methods（χ²＝2.952, P＝0.086). The spectrograms of 4 isolates with identification score less than 1.700 were filled of impurities and lack of valid peaks. Conclusion：The improved direct MALDI-TOF MS method（"Tongren") established by our lab for the identification of mycobacteria from liquid media may obtain accurate results, shorten the culture and identification time of mycobacteria, and ensure biological safety, which may be helpful for the rapid diagnosis and treatment of mycobacteria infections, and be applied to different levels of clinical laboratories.