Objective: To evaluate the levels of neuron-specific enolase (NSE), carcinoembryonic antigen (CEA) and pro-gastrin releasing peptide (pro-GRP) in the serum of patients with lung cancer and analyze clinical significance of NSE/CEA and pro-GRP/CEA ratio in the differential diagnosis of small cell lung cancer (SCLC). Methods: Forty-five cases with SCLC and 60 patients with non-small cell lung cancer lung cancer (NSCLC), including 22 cases with lung squamous carcinoma and 38 cases with lung adenocarcinoma were selected. The levels of multiple serum tumor markers (NSE, CEA, pro-GRP) in the serum of patients were detected by electrochemiluminescence immunoassay. And the ratio of NSE/CEA, pro-GRP/CEA were calculated. ROC curve was used to evaluate the best value of NSE/CEA and pro-GRP/CEA ratio in the differential diagnosis of SCLC. Results: At the threshold of 3.78, sensitivity and specificity of NSE/CEA as a serologic marker were 88.90% and 85.00%, respectively for distinguish SCLC and NSCLC. ROC curve showed that area under the curve (AUC) was 0.90. At the threshold of 28.61, sensitivity and specificity of pro-GRP/CEA as a serologic marker were 86.70% and 91.70%, respectively for distinguish SCLC and NSCLC. ROC curve showed that AUC was 0.94. Conclusion: Combined examination of multiple serum tumor markers and the NSE/CEA and pro-GRP/CEA ratio can effectively improve the accuracy in the differential diagnosis of SCLC.