Objective: To evaluate the diagnostic value of tuberculosis (TB) and drug-resistant gene rapid detection (TB-Gene Xpert MTB/RIF) for laryngeal tuberculosis. Methods: A retrospective analysis was conducted on 94 suspected laryngeal tuberculosis patients who received initial diagnosis at Renmin Hospital of Wuhan University from January 2018 to June 2024. All patients underwent chest CT examination, blood TB infection T-cell detection (T-spot), and sputum smear acid-fast staining. Tissue samples were collected under electronic laryngoscopy for TB-Gene Xpert MTB/RIF detection, pathological examination, and TB culture. The diagnostic efficacy of chest CT, T-spot, sputum smear, tissue TB culture, pathological examination, and TB-Gene Xpert MTB/RIF in the diagnosis of laryngeal tuberculosis was evaluated using clinically confirmed laryngeal tuberculosis patients as the gold standard. Results: A total of 94 suspected laryngeal tuberculosis patients were included in this study, with 59 confirmed cases of laryngeal tuberculosis and 35 non-laryngeal tuberculosis cases. Referencing the 59 confirmed cases of laryngeal tuberculosis, the sensitivities of chest CT, T-spot, sputum smear, tissue culture, pathological examination, and TB-Gene Xpert MTB/RIF were 59.3% (35/59), 67.8% (40/59), 25.4% (15/59), 35.6% (21/59), 79.7% (47/59), and 100% (59/59), respectively; their specificities were 42.9% (15/35), 51.4% (18/35), 91.4% (32/35), 100% (35/35), 85.7% (30/35), and 94.3% (33/35), respectively. The sensitivity of TB-Gene Xpert MTB/RIF detection was significantly higher than other detection methods (P<0.01), and its specificity was significantly higher than chest imaging and T-spot (P<0.01), with no significant difference in specificity compared to sputum smear, tissue culture, and pathological examination (P>0.05). Two rifampicin-resistant strains were detected in patients with laryngeal tuberculosis, and TB-Gene Xpert MTB/RIF detected rpoB gene mutations with a sensitivity of 100%. All laryngeal tuberculosis patients had a good prognosis after receiving systemic anti-tuberculosis treatment. Conclusion: The TB-Gene Xpert MTB/RIF technology has the advantages of being rapid, simple, highly sensitive, and highly specific in the diagnosis of laryngeal tuberculosis. It can effectively identify rifampicin-resistant patients and is suitable for early diagnosis of laryngeal tuberculosis, helping to reduce the risk of missed and misdiagnoses.