The role of serum CA125 and CEA in the prognosis of non-small cell lung cancer (NSCLC) remains controversial, especially in early stage patients, which need further clarification. Thus, in this study we carried out a large scale retrospective analysis of the prognostic value of CA125 and CEA in 645 patients with NSCLC, to explore their predictive value in the NSCLC. Patients who underwent curative surgical resection for NSCLC were from Zhejiang Cancer Hospital of China from 2006 to 2011. Microparticle enzyme immunoassay was used to measure preoperative serum CA125 and CEA. Univariate analyses and a multivariable proportional hazard Cox regression model were applied to assess the prognostic significance of the different covariates. Kaplan-Meier method was used to analyze survival curve. Both CA125 and CEA were correlated with stage, but also CA125 was different by grade, and CEA was related to histology. The Kaplan-Meier survival analysis showed that patients with elevated CA125 or CEA had unfavorable disease progression-free and overall survival time compared to those with normal CA125 or CEA. Furthermore, multivariate Cox analysis revealed that elevated CA125 had significantly higher risk for relapse (HR, 1.76; p=0.001) and death (HR, 1.80; p<0.001), but not for elevated CEA as relapse (HR, 1.06; p=0.736) and death (HR, 1.25; p= 0.119) both were statistically non significant. This study showed that both CA125 and CEA play important roles in disease progression while only CA125 as an independent predictive marker for prognosis in patients with NSCLC.