Background: Non-small cell lung (NSCLC) holds high mortality owing to the difficulty to early detection from other lung mass, such as tuberculosis. This study evaluates the clinical value of the combination of circulating cell-free DNA (cfDNA) quantification and metabolic tumor burden to distinguish NSCLC from tuberculosis. Methods: A total of 149 NSCLC patients, 151 tuberculosis patients and 150 healthy controls were included. Quantifying serum cfDNA fragments from ALU (115 bp) gene by RT-PCR. Metabolic tumor burden (SUV-Maxa) values were detected by preoperative the 18F-fluorodeoxyglucose positron emission tomography (18F-FDG PET/CT). A549 cell, NCI–H460 cell, NSCLC and tuberculosis mice model were used to elucidate the specific mechanism. Results: Serum cfDNA levels and SUV-Maxa were higher in NSCLC patients than those in healthy controls and those in tuberculosis. Meanwhile, mice models showed the similar discovery. In addition, obvious correlations of cfDNA and metabolic tumor burden were only existed in NSCLC patients and mice model, rather than tuberculosis and control. Moreover, the combination of cfDNA and metabolic tumor burden displayed better effect to distinguish NSCLC from tuberculosis than alone use. Mechanistically, upregulated Glucose transporter 1 (GLU1) increased necroptosis-induce cfDNA rise by FasL/caspase 8/caspase 3 pathway and promoted metabolic tumor burden in NSCLC. Conclusions: The combination of cfDNA and metabolic tumor burden displayed better effect to distinguish NSCLC from tuberculosis, owing to upregulated GLU1 increased cfDNA levels by FasL/caspase 8/caspase 3 pathways and promoted metabolic tumor burden in NSCLC.