COVID-19 disease has led to many deaths worldwide and early detection of people at a high risk of severe forms of this disease would greatly help physicians. The presence of oxidative stress biomarkers may help identify high-risk individuals early in the course of the disease. 8-Hydroxy-2'-deoxyguanosine (8-OHdG) is a widely used biomarker for assessing endogenous oxidative DNA damage. In this study, the urinary 8-OHdG levels were determined in COVID-19 patients and COVID-19 patients with cancer by a dispersive liquid-liquid microextraction (DLLME) method using gas chromatography-mass spectrometry (GC-MS). The effects of essential parameters on the extraction method were investigated. The LOD and LOQ are equal to 1.7 nM and 5.1 nM, respectively. At varied concentrations of 8-OHdG (300, 400, and 600 nM), the relative standard deviation (RSD) ranged from 18.35% to 22.36%. The mean urinary 8-OHdG levels of cancer and COVID-19 patients were 13.20 ± 6.20 nmol mmol-1, while the mean levels in COVID-19 patients and healthy volunteers were 6.67 ± 5.80 nmol mmol-1 and 1.61 ± 1.72 nmol mmol-1, respectively. The results of this study showed that the level of 8-OHdG urine biomarkers in people with COVID-19 is significantly higher than in healthy people. In this study, the DLLME approach was used for the first time to determine the value of 8-OHdG using GC-MS. According to the results of this research, the DLLME method was successfully used as a biomarker of DNA oxidative stress for extracting 8-OHdG urine. Compared to other methods, this technique has advantages such as shorter extraction time and low cost.