Background: As a minimally invasive method, endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) was more accurate than non-invasive methods such as positron emission tomography (PET) and computed tomography (CT) to evaluate the lymph nodes in preoperative non-small cell lung cancer (NSCLC).PET/CT has more anatomical advantages than PET scanning and is more accurate in lung cancer staging. However, no relevant studies have comparatively evaluated PET/CT and EBUS-TBNA for NSCLC patients.Methods: A total of 112 patients were included in this retrospective analysis. The golden diagnosis of N2 status was postoperative pathological results. In EBUS-TBNA puncture specimens, if clear malignant tumor cells could be seen, the results were taken as positive. In PET/CT image analysis, the CT values, short diameter, and maximum standardized uptake value (SUVmax) of each lymph node were recorded to evaluate N2 status. The results of PET/CT and EBUS-TBNA were compared with the final pathological results, and respective sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were calculated. -Then, the patients were divided into adenocarcinoma group and squamous cell carcinoma group -and the results were calculated and compared with the above method. Results:The results showed that EBUS-TBNA had a higher diagnostic value for mediastinal lymph nodes than PET/CT, and the difference was statistically significant (P<0.001). In NSCLC patients, the results showed that the sensitivity (P=0.013), specificity (P<0.001), PPV (P<0.001), NPV (P<0.001), and accuracy (P<0.001) of EBUS-TBNA were higher than that of PET/CT (AUC =0.954 and 0.636, respectively).In adenocarcinoma cases, specificity (P<0.001), PPV (P<0.001), NPV (P<0.001), and accuracy (P<0.001) of EBUS-TBNA were higher than that of PET/CT (AUC =0.957 and 0.596, respectively).In cases with squamous cell carcinoma, specificity (P=0.003), PPV (P<0.001), and accuracy (P<0.001) of EBUS-TBNA were higher than PET/CT (AUC =0.952 and 0.657, respectively).Conclusions: For preoperative diagnosis of mediastinal lymph node metastases in NSCLC, EBUS-TBNA is more accurate than PET/CT. For those patients with suspected mediastinal lymph node metastasis, EBUS-TBNA should be preferred method to evaluate the status of mediastinal lymph nodes.