Following accidental radiation exposure due to a radiological or nuclear emergency, a dose assessment should be performed based on biological samples from exposed individuals. Although previous biological dose assessment approaches have focused on nuclear DNA damage in peripheral lymphocytes, this study investigated the radiation-induced impact on the mitochondrial genome, particularly the chronological changes in the mitochondrial DNA copy number (mtDNAcn) following exposure to radiation. We used B-lymphoblastoid cell lines transformed by Epstein-Barr virus established from 12 healthy individuals in their 20s (six males, six females) with or without a history of smoking. Using real-time quantitative PCR, we determined the mtDNAcn from the B cells cultured for 6, 24, and 96 h and after exposure to 0, 1, 2, and 4 Gy X-rays. We found a significant relationship between the exposed dose and mtDNAcn in the 96-hour post-irradiation cells for non-smoking males, suggesting the possible role of mtDNAcn as a biomarker for dose assessment.