Abstract Background Analysis of free DNA molecules shed from tumour cells in plasma of patients referred as circulating tumour DNA (ctDNA) with reference to physiological circulating cell-free DNA (cfDNA) is nowadays exploited as liquid biopsy and is considered a new emerging promising biomarker for diagnosis, selection of proper treatment, and prognosis of cancer. DNA integrity index (DII) is assessed by calculating the ratio between the concentration of long cfDNA strands released from tumour cells (ALU247) and the short strands released from normal cells (ALU115). The aim of the current study was to evaluate DII as a potential diagnostic and prognostic biomarker of NSCLC. Methods Our study included 48 NSCLC patients diagnosed as primary NSCLC before starting treatment, 30 COPD patients diagnosed clinically, radiologically, and subjected to chest high-resolution computerized tomography, and 40 healthy controls. cfDNA concentration and DII were measured by quantitative real-time polymerase chain reaction (qPCR). Results ALU115, ALU247, and DII were significantly higher in NSCLC compared to COPD patients (p