BACKGROUND Lung adenosquamous carcinoma (ASC) is a rare malignant tumor with an adenocarcinoma and a squamous cell carcinoma component and associated with a lower 5-year survival rate than lung squamous cell carcinoma and lung adenocarcinoma. Surgical specimen histology revealed the inadequacy of conventional transbronchial needle aspiration samples in the diagnosis of lung ASC. Most lung ASC patients are not suitable to receive surgery, and it is difficult to diagnose ASC. This study is to explore the possibility of using serum carcinoembryonic antigen (CEA) and squamous cell carcinoma antigen (SCC) as a supplementary diagnostic test for ASC. METHODS We retrospectively analyzed the preoperative serum CEA and SCC levels in 34 patients with lung ASC, 35 cases of lung adenocarcinoma patients, 35 cases of lung squamous cell carcinoma patients. 36 cases of lung benign disease patients and 35 cases of healthy people as a control group were also retrospectively collected and analyzed from January 2012 to December 2014 at the Zhejiang Cancer Hospital, China. The differences of CEA and SCC among the groups were evaluated, and the area under the curve (AUC), sensitivity, and specificity were calculated. RESULTS The levels of SCC and CEA in the lung ASC group were significantly higher than those in the healthy control group and benign disease group (p < 0.05). The SCC level in lung ASC group was significantly higher than that in lung adenocarcinoma group (p < 0.05). CEA and SCC had good diagnostic sensitivity and specificity compared with the healthy control group, and the difference was statistically significant (p < 0.05). CONCLUSIONS Our retrospective study suggested a role for serum CEA and SCC levels as reference markers in the diagnosis of lung ASC. Patients with elevated CEA and SCC levels and diagnosed as lung adenocarcinoma by limited biopsy materials should be offered further work-up to reach an accurate diagnosis and treatment.