Objective To analyze the relationship between carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125), neuron-specific enolase (NSE) and the pathological characteristics of patients with non-small cell lung cancer (NSCLC) and the value of combined detection. Methods Totally 175 NSCLC patients (NSCLC group) who were treated in Shanghai Jinshan District Hospital of Integrated Traditional Chinese and Western Medicine (January 2018-January 2022), and 180 patients with benign pulmonary diseases during the same period (no-NSCLC group) were retrospectively enrolled. The clinical data, the level of CEA, CA125, and NSE of patients at different stages were included as comparators. Spearman correlation analysis was conducted to assess the association between serum indexes and staging system. the diagnostic value of CEA, CA125 and NSE of NSCLC patients, alone and in combination, were analyzed by the receiver operating characteristic (ROC) curve.Results The serum levels of CEA, CA125 and NSE in NSCLC group were markedly higher than those in no-NSCLC group ( <0.05). The significant increasing trend for serum levels of CEA, CA125 and NSE were found in patients with classification grades Ⅰ-Ⅲ( <0.05). The results of Spearman correlation analysis showed that CEA, CA125, NSE were positively correlated with the clinical stage of patients with NSCLC ( =0.579, 0.437, 0.686, <0.05). The ROC curve was drawn with NSCLC as positive and benign lung disease as negative, the area under the curve (AUC) of CEA, CA125, and NSE for predicting NSCLC, alone and in combination, were 0.809, 0.842, 0.867, and 0.910, respectively ( <0.05). Conclusion The increased expressions of CEA, CA125 and NSE are detected in NSCLC patients, and increasing trend with the disease progression, the detection of serum levels of CEA, CA125 and NSE alone and in combination had certain diagnostic value for NSCLC.