Loss of 14-3-3σ expression through DNA methylation has been associated with carcinogenesis and the prognosis for various cancer types. Detection of methylation of the gene in serum may be useful for diagnostic utility. The present study aimed to investigate the correlation between 14-3-3σ methylation level in 36 paired tumor tissues of non-small cell lung cancer (NSCLC) and matched serum using methylation-specific polymerase chain reaction. The prognostic significance of 14-3-3σ expression in 167 NSCLC was also evaluated using immunohistochemistry. Methylation of the 14-3-3σ gene was identified in all samples. The methylation level in the serum (mean 87.7%, range 64.6–100%) was higher compared with tumor (mean 46.7%, range 25.3–56.3%). However, no significant correlation between methylation levels in tissues and serums was observed (Spearman's correlation, −0.036; P=0.837). In the 167 tumor tissues, the majority of the cases (83.8%) exhibited negative expression. Adenocarcinoma is more likely to exhibit negative expression (91.4%) compared with squamous cell carcinoma (70.2%). No significant difference was identified in the overall survival according to 14-3-3σ expression status and 14-3-3σ expression did not demonstrated independent prognostic significance. In conclusion, NSCLC harbors certain levels of 14-3-3σ methylation in the tumor and the sera of patients. The clinical value of serum 14-3-3σ methylation should be further elucidated. Immunohistochemical expression 14-3-3σ protein has limited value on prognostic significance.