Lung cancer remains a global health problem with a high mortality rate. CpG island methylation is a common aberration frequently associated with gene silencing in multiple tumor types, emerging as a highly promising biomarker. The trans-membrane protein with a single EGF-like and two follistatin domains (TMEFF2) is epigenetically silenced in numerous tumor types, suggesting a potential role as a potential tumor suppressor. However, the role of TMEFF2 in lung cancer remains to be fully elucidated. We explored the methylation status of the TMEFF2 gene in 139 patients with non-small cell lung cancer (NSCLC) and the feasibility of detecting circulating methylated DNA as a screening tool for NSCLC using methylation-specific PCR (MSP). TMEFF2 methylation in tumor tissues was found in 73 of the 139 NSCLCs (52.5%) and was related to gene expression. In adenocarcinomas, TMEFF2 methylation was significantly more frequent in tumors without EGFR mutation than those with EGFR mutation (adjusted hazard ratio=, 95% confidence interval=, P=0.002). In addition, the frequency of TMEFF2 methylation was also higher in females and never-smokers than in males and smokers with borderline significance (65.8% vs 47.8%, p=0.06; 65.7% vs 48.1%, p=0.07). Furthermore, TMEFF2 methylation was exclusively detected in the serum of NSCLC patients at a frequency of 9.2% (29/316). These findings suggest that the methylation-associated down-regulation of the TMEFF2 gene may be involved in early lung tumorigenesis and TMEFF2 methylation can serve as a specific blood-based biomarker for NSCLC.