Objective To investigate change in amount of serum cell-free mitochondrial DNA (cf-mtDNA) related with inflammatory response and to explore clinical relevance to trauma patients. Methods Thirty-seven trauma patients as trauma group were divided as multiple trauma subgroup or single trauma subgroup, and as shock subgroup or non-shock subgroup. Ten healthy volunteers were as control group. The amount of cf-mtDNA in serum at 4 h, 24 h, 72 h, 7 d post-injury was compared between trauma group and control group and among subgroups. The association of serum cf-mtDNA with injury severity score (ISS), systemic inflammatory response syndrome (SIRS) score, C-reactive protein (CRP), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were analyzed respectively. The receiver operating characteristic (ROC) curve of serum cf-mtDNA post-injury were constructed to evaluate role in diagnosing SIRS. Results Serum cf-mtDNA in trauma patients at 24 h, 72 h post-injury were significantly higher than those in control group (P<0.05). Serum cf-mtDNA of patients in multiple trauma subgroup were significantly higher than those in single trauma subgroup at 4 h, 24 h, 72 h, 7 d post-injury (P<0.05, P<0.01, P<0.01, P<0.05). The amount of serum cf-mtDNA in shock subgroup were significantly higher than that in non-shock subgroup at 24 h post-injury (P<0.01), and that in group of organ dysfunction higher than in group without organ dysfunction at 4 h, 24 h post-injury (P<0.05). The peak of serum cf-mtDNA in dead patients was hi-gher than survived patients without statistical significance (P>0.05). Amounts of TNF-α, IL-6 and CRP in serum increased with serum cf-mtDNA post-injury. Amount of serum cf-mtDNA correlated positively with ISS at 24 h post-injury (r=0.454, P=0.004), with SIRS score and serum IL-6 at 4 h-7 d post-injury respectively(r=0.458, P=0.000 1; r=0.252, P=0.005), but did not correlate with serum TNF-α (r=-0.058, P=0.511), and with CRP at 4-24 h post-injury positively (r=0.264, P=0.028). The area under ROC curve of serum cf-mtDNA was 0.752 at 4 h-7 d post-injury (P=0.000 01), with 95% confidence interval of 0.668-0.836. According to maximum Youden index, the optimal cutoff value of serum cf-mtDNA was 0.075 2 with the sensitivity 63.6% and specificity 85.5% for diagnosis of SIRS. Conclusions The amount of serum cf-mtDNA increased in trauma patients at early stage, indicating severe trauma and inflammation, and more amount of serum cf-mtDNA would be helpful to diagnose SIRS.