BACKGROUND Fractional exhaled nitric oxide (FeNO) is an easy, sensitive, reproducible, and noninvasive marker of eosinophilic airway inflammation. Accordingly, FeNO is extensively used to diagnose and manage asthma. Patients with COPD who share some of the features of asthma have a condition called asthma-COPD overlap syndrome (ACOS). The feasibility of using FeNO to differentiate ACOS patients from asthma and COPD patients remains unclear. METHODS From February 2013 to May 2016, patients suspected with asthma and COPD through physician's opinion were subjected to FeNO measurement, pulmonary function test (PFT), and bronchial hyperresponsiveness or bronchodilator test. Patients were divided into asthma alone group, COPD alone group, and ACOS group according to a clinical history, PFT values, and bronchial hyperresponsiveness or bronchodilator test. Receiver operating characteristic (ROC) curves were obtained to elucidate the clinical functions of FeNO in diagnosing ACOS. The optimal operating point was also determined. RESULTS A total of 689 patients were enrolled in this study: 500 had asthma, 132 had COPD, and 57 had ACOS. The FeNO value in patients with ACOS was 27 (21.5) parts per billion (ppb; median [interquartile range]), which was significantly higher than that in the COPD group (18  ppb). The area under the ROC curve was estimated to be 0.783 for FeNO. Results also revealed an optimal cutoff value of >22.5 ppb FeNO for differentiating ACOS from COPD patients (sensitivity 70%, specificity 75%). CONCLUSION FeNO measurement is an easy, noninvasive, and sensitive method for differentiating ACOS from COPD. This technique is a new perspective for the management of COPD patients.