18F-Alfatide II has been translated into clinical use and been proven to have good performance in identifying breast cancer. In this study, we investigated 18F-Alfatide II for evaluation of axillary lymph nodes (ALN) in breast cancer patients and compared the performances with 18F-FDG. Methods: A total of 44 female patients with clinically suspected breast cancer were enrolled and underwent 18F-Alfatide II and 18F-FDG PET/CT within a week. Tracer uptakes in ALN were evaluated by visual analysis, semiquantitative analysis with maximum standardized uptake value (SUVmax), mean standardized uptake value (SUVmean) and SUVmax ratio of target/non-target (T/NT). Results: Among 44 patients, 37 patients were pathologically diagnosed with breast cancer with metastatic (17 cases) or non-metastatic (20 cases) ALN. The sensitivity, specificity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) of visual analysis were 70.59%, 90.00%, 81.08%, 85.71% and 78.26% for 18F-Alfatide II, 64.71%, 90.00%, 78.38%, 84.62% and 75.00% for 18F-FDG, respectively. By combining 18F-Alfatide II and 18F-FDG, the sensitivity significantly increased to 82.35%, the specificity was 85.00%, the accuracy increased to 83.78%, the PPV was 82.35% and the NPV significantly increased to 85.00%. Three cases of luminal B subtype were false negative for both 18F-Alfatide II and 18F-FDG. The other 2 false negative cases of 18F-Alfatide II were triple-negative subtype and 3 false negative cases of 18F-FDG were luminal B subtype too. The AUCs of three semi-quantitative parameters (SUVmax, SUVmean, T/NT) for 18F-Alfatide II were between 0.8 and 0.9, whereas those for 18F-FDG were more than 0.9. 18F-Alfatide II T/NT had the highest Youden index (76.5%), specificity (100%), accuracy (89.19%) and PPV (100%) among these semi-quantitative parameters. 18F-Alfatide II uptake as well as 18F-FDG uptake in metastatic axillary lymph nodes (MALN) was significantly higher than that in benign axillary lymph nodes (BALN). Both 18F-Alfatide II and 18F-FDG did not show difference in primary tumor uptake irrespective of ALN status. Conclusion: 18F-Alfatide II can be used in breast cancer patients to detect metastatic ALN, however, like 18F-FDG, with high specificity but relatively low sensitivity. The combination of 18F-Alfatide II and 18F-FDG can significantly improve sensitivity and NPV. 18F-Alfatide II T/NT may serve as the most important semi-quantitative parameter to evaluate ALN.