BACKGROUND The association between mitochondrial DNA (mtDNA) copy number and head and neck squamous cell carcinoma (HNSCC) risk remains unclear. Therefore, we aimed to evaluate the relationship between mtDNA copy number and HNSCC risk. MATERIAL AND METHODS We searched PubMed, Web of Science, and EMBASE until August 2020. Studies that assessed the association between mtDNA copy number and HNSCC as the outcome of interest were included. We performed a 2-class and dose-response meta-analysis to assess the association between cancer risk and mtDNA. RESULTS Eight articles (2 cohort studies and 6 case-control studies) with a total of 3913 patients were included in our meta-analysis. The overall results showed that mean mtDNA copy number level from 9 studies was 0.71 higher in patients with cancer than in non-cancer controls (the standardized mean differences (SMD) 0.71, 95% CI: 0.28-1.15, P<0.001). However, when 4 studies were pooled by dichotomizing mtDNA copy number at the median value into high- and low-content groups, no significant association between mtDNA content and overall cancer risk was found (odds ratio (OR)=0.87, 95% CI: 0.52-1.44, P=0.584). Furthermore, we observed a non-linear association from 3 studies between increased mtDNA copy number levels (P for nonlinearity <0.001). CONCLUSIONS The elevated mtDNA copy number could predict the risk of HNSCC as a biomarker. Moreover, there was non-linear relationship of risk between HNSCC and mtDNA copy number.