The aim of the study was to investigate the clinical significance of serum mitochondrial DNA (mtDNA) in lung cancer.Serum mtDNA from 65 lung cancer patients, 20 patients with benign lung diseases and 55 healthy individuals was quantified using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR). Data were analyzed using statistical software SPSS 13.0.Serum mtDNA levels in lung cancer patients were significantly higher, compared to those in patients with benign lung diseases and healthy individuals (u=108, p=0.000; u=293, p=0.000), and closely associated with TNM stage (p=0.01). The use of serum mtDNA facilitated detection of lung cancer at a cutoff value of 0.74×10⁴ copies/μL with a sensitivity of 86.2% and specificity of 80.7%. However, serum mtDNA levels were not associated with patient age, gender, histological type, and lymph node metastasis (p>0.05).Quantification of serum mtDNA using FQ-PCR potentially serves as a novel complementary tool to improve the clinical screening and detection of lung cancer.