Mitochondrial (mt) DNA has been long suggested to contribute to carcinogenesis, and a variety of mutations in mtDNA have been confirmed to be related to various early stages of cancers; these data revealed that the detection of mtDNA in clinical samples may be a promising approach for cancer diagnosis. In the present study, the serum mtDNA in healthy donors and groups of patients with cancer was detected. It was revealed that patients with lung cancer without metastasis had more mtDNA in serum compared to patients with metastasis. Moreover, TLR9‑associated signalling was activated in vitro after treatment with a synthetic CpG oligodeoxyribonucleotide (ODN) called ODN‑M362. In addition, our data revealed that TLR9 and its adaptor protein, MyD88, were induced by ODN‑M362 in a dose‑dependent manner. A human cytokine array to evaluate stimulation of cytokine secretion by ODN‑M362 was also used. Our findings may identify the role that TLR9 and mtDNA play in lung cancer progression and metastasis.