The objectives of this study were to compare the diagnostic potential of 68 Ga-Alfatide II with 18 F-FDG in differentiating between non-small cell lung cancer patients (NSCLC) and lung tuberculosis (TB) patients. Methods: Twenty-one NSCLC patients and 13 TB patients were recruited. PET/CT images using either 68 Ga-Alfatide II or 18 F-FDG were acquired in 2 consecutive days. SUV quantitative comparison, receiver-operating curve analysis, and comprehensive visual analysis were performed. The expression of the angiogenesis marker α v β 3 in NSCLC and TB primary lesions was analyzed by immunohistochemistry. Results: The 68 Ga-Alfatide II SUV max and SUV mean were significantly different in NSCLC and TB (P 5 0.0001 and 0.0007, respectively). The area under the receiver-operating curve value of 68 Ga-Alfatide II SUV max was significantly higher than that of 18 F-FDG (P 5 0.038). The visual differentiation diagnostic specificity of 68 Ga-Alfatide II was 1.57-fold (84.62% vs. 53.85%) higher than that of 18 F-FDG. In the detection of NSCLC lymph nodes, 68 Ga-Alfatide II was superior in specificity (100% vs. 66.7%), whereas the sensitivity was greater with 18 F-FDG (87.5% vs. 75%). In TB lymph node detection, the false-positive rate of 68 Ga-Alfatide II was one-third (15.4%/ 46.2%) the value of 18 F-FDG. Additionally, 68 Ga-Alfatide II detected more metastases in the brain but less in the liver and the bone. The α v β 3 biomarker was specifically expressed in the cells and the neovasculature of NSCLC lesions. Conclusion: 68 Ga-Alfatide II is qualified for detecting NSCLC primary lesions and is superior to 18 F-FDG in distinguishing NSCLC from TB in primary lesions and suggestive lymph nodes. 68 Ga-Alfatide II is more likely to be capable of detecting brain metastasis, and 18 F-FDG is more likely to be capable of detecting liver and early-stage bone metastases.