Smoking exposes individuals to a range of harmful chemicals linked to various diseases and remains one of the most common unhealthy behaviors. The objectives of this research are to investigate the correlation between smoking and mtDNA-CN using measured levels of Interleukin 6 (IL-6) and High-Sensitivity C-Reactive Protein (hs-CRP) as an inflammatory response. The current study included 80 smokers and 60 non-smokers. The levels of IL-6 and hs-CRP were measured using the ELISA method, and mtDNA-CN was measured using quantitative real-time PCR. Our study showed that the relative mtDNA/nDNA ratio was lower in smokers compared to non-smokers (0.8186 vs. 1.740; p = 0.0001). The AUC value was 0.8442 (p < 0.0001). We also found that mtDNA-CN was inversely correlated with IL6 (r = -0.5867, p < 0.0001), and hs-CRP (r= -0.4668, p < 0.0001). The mtDNA-CN could potentially serve as a predictive biomarker for the impact of smoking on human health and diseases.