Our study aims to determine the clinical significance of human telomerase reverse transcriptase (hTERT), S-phase kinase-associated protein 2 (Skp2) and thyroid transcription factor-1 (TTF-1) mRNA expressions in peripheral blood (PB) of patients with non-small cell lung cancer (NSCLC). Real-time polymerase chain reaction was used to investigate the gene expressions of hTERT, Skp2, TTF-1 as in the PB of 60 patients with NSCLC and 20 benign lung diseases. Statistical analyses were performed to examine the correlation between the expression of these mRNA markers and the clinical pathological features of NSCLC. We found that hTERT, Skp2, and TTF-1 were overexpressed in the PB of NSCLC patients, and demonstrated high specificity as well as sensitivity when used for NSCLC diagnosis. Significant correlation was observed between disease stage and the three markers (P < 0.05). This study suggests that the genes hTERT, Skp2, and TTF-1 play important roles in tumor genesis and development, and can be used as diagnosis markers in NSCLC patients. The expression of three markers in combination can significantly improve the sensitivity and accuracy of diagnosis relative to single marker diagnosis, and provides a reliable method to detect CTCs in the PB. Additionally, these markers can also be used as diagnostic markers for clinical stages of NSCLC.