Objective: To explore the application value of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) specimens combined with DNA methylation detection in the clinical diagnosis of lung cancer, compare the diagnostic efficacy of single detection methods, and provide objective evidence for early and accurate diagnosis of lung cancer. Methods: A total of 120 patients with suspected pulmonary malignancies admitted to our hospital from December 2024 to December 2025 were selected. All patients underwent EBUS-TBNA to obtain biopsy specimens, and routine pathological cytological examination, individual detection of SHOX2 and RASSF1A gene methylation, and combined detection of both methods were performed. Using postoperative pathology or long-term follow-up results as the gold standard, the sensitivity, specificity, accuracy, positive predictive value, negative predictive value, and area under the ROC curve (AUC) of each detection method were calculated, and diagnostic differences were compared. Results: Among the 120 patients, 76 cases (63.33%) were diagnosed with lung cancer and 44 cases (36.67%) with benign lesions according to the gold standard. The sensitivity, specificity, and accuracy of routine cytological examination by EBUS-TBNA were 72.37%, 84.09%, and 76.67%, respectively; for individual DNA methylation detection, they were 78.95%, 86.36%, and 81.67%, respectively; for combined detection, they were 93.42%, 95.45%, and 94.17%, respectively. All diagnostic indicators of combined detection were significantly superior to those of single detection methods (P < 0.05). The area under the ROC curve for combined detection was 0.980, significantly higher than that for individual cytological detection (0.908) and individual methylation detection (0.934). Conclusion: EBUS-TBNA specimens combined with DNA methylation detection can significantly improve the sensitivity and accuracy of lung cancer diagnosis, overcome the limitations of routine cytological examination, and are particularly suitable for difficult cases such as mediastinal lymphadenopathy and peripheral pulmonary nodules, demonstrating high clinical promotion value.