Sex-determining region Y-box 2 (SOX2), as a subunit of transcription and reprogramming factor, plays a critical role in the development and progression of many malignancies, including lung cancer through gene amplification. In the present study, we aimed to quantify the levels of serum SOX2 DNA, analyze its diagnostic value and compare it with existing clinical parameters in lung cancer, and purpose to provide a novel tumor marker for lung cancer. Serum DNA was extracted from 94 lung cancer patients, 10 benign lung diseases, and 30 healthy volunteers, and then the levels of SOX2 DNA were quantified using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR). The data were analyzed by statistical software SPSS14.0. The present results show that serum SOX2 DNA level in lung cancer group was higher compared to the levels in benign lung diseases group (u = 102.0, p < 0.001) or healthy group (u = 140.0, p < 0.001), and it was closely associated with TNM stage, histopathological type, and tumor size (p = 0.031, p = 0.012, and p = 0.010, respectively). However, serum SOX2 DNA levels of lung cancer patients were not associated with age, gender, smoking status, lymph node metastasis, or tumor differentiation (p > 0.05). ROC curve showed a sensitivity of 78.9% and a specificity of 82.5% for the ability of serum SOX2 DNA to detect lung cancer at the cutoff value of 1,078.3 copies/ul. Furthermore, we assessed the associations of serum SOX2 levels with clinical existing lung tumor markers, such as squamous cell carcinoma antigen, cytokeratin fragment 21-1, and neuron-specific enolase. The sensitivity was increased from 24.9, 66.1, and 39.1 to 84.2, 92.8, and 87.5%, respectively, by the combination of serum SOX2 DNA. Taken together, quantification of serum SOX2 DNA by FQ-PCR may serve as a novel accessory diagnostic tool for the clinical screening and detection of lung cancer.