AbstractAcetaminophen (APAP) overdose is a leading cause of drug‐induced liver injury and acute liver failure, while the detection, prognosis prediction, and therapy for APAP‐induced liver injury (AILI) remain improved. Here, it is determined that the temporal pattern of circulating cell‐free DNA (cfDNA) is strongly associated with damage and inflammation parameters in AILI. CfDNA is comparable to alanine aminotransferase (ALT) in predicting mortality and outperformed ALT when combined with ALT in AILI. The depletion of cfDNA or neutrophils alleviates liver damage, while the addition of cfDNA or adoptive transfer of neutrophils exacerbates the damage. The combination of DNase I andN‐acetylcysteine attenuates AILI significantly. This study establishes that cfDNA is a mechanistic biomarker to predict mortality in AILI mice. The combination of scavenging cfDNA and reducing oxidative damage provides a promising treatment for AILI.