The clinical phenotypes and underlying mechanisms of asthma-COPD overlap syndrome (ACOS) remain elusive. This study aimed to investigate a comparison of COPD patients with and without ACOS, focusing on inflammatory biomarkers, in an outpatient COPD cohort.We conducted a cross-sectional study analyzing prospectively collected data from the Ishinomaki COPD Network registry. All participants were diagnosed with COPD, confirmed by using spirometry, and were aged 40-90 years and former smokers. Patients with features of asthma including both variable respiratory symptoms and variable expiratory airflow limitation were identified and defined as having ACOS. Then, the inflammatory biomarkers such as fractional exhaled nitric oxide level, blood eosinophil count and percentage, total immunoglobulin E (IgE) level, and presence of antigen-specific IgE were evaluated.A total of 257 patients with COPD were identified, including 37 (14.4%) with ACOS. Patients with ACOS tended to be younger, have a shorter smoking history, and use more respiratory medications, especially inhaled corticosteroids and theophylline. Mean fractional exhaled nitric oxide level was significantly higher in those with ACOS than in those without ACOS (38.5 parts per billion [ppb] vs 20.3 ppb, P<0.001). Blood eosinophil count and percentage were significantly increased in those with ACOS (295/mm3 vs 212/mm3, P=0.032; 4.7% vs 3.2%, P=0.003, respectively). Total IgE level was also significantly higher, and presence of antigen-specific IgE was observed more frequently in patients with ACOS. Receiver operating characteristic curve analysis indicated that the sensitivity and specificity of these biomarkers were relatively low, but combinations of these biomarkers showed high specificity for ACOS diagnosis.These results provide evidence that these inflammatory biomarkers can be used to support the diagnosis of ACOS.