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The diagnosis of tuberculous mediastinal lymphadenitis remains a challenge, and the use of transbronchoscopic needle aspiration (TBNA) combined with rapid on-site cytological evaluation (ROSE) is still unclear. A case of tuberculous mediastinal lymphadenitis was illustrated to show the diagnostic value of TBNA and ROSE.In this case report, we presented a typical case of a 44-year-old male who underwent obvious odynophagia and mild symptom of dyspnea. One isolated mass positioned on posterior mediastinum was examined as positive discovery. Finally, he was diagnosed with tuberculous mediastinal lymphadenitis by using TBNA combined with ROSE and treated with anti-TB.TBNA is an efficacious and safe approach, which is worth popularizing for the clinical diagnosis of mediastinal masses. Meanwhile, ROSE is useful to reduce the numbers of needle passes during TBNA. We aimed to emphasize 2 key points in this case report. Firstly, a rare symptom of Tuberculosis in adults was supported by the patient. Secondly, TBNA combined with ROSE is useful for the diagnosis of tuberculous mediastinal lymphadenitis.