Objectives To evaluate accurately hepatocyte injury degree of severe hepatitis patients by quantifying plasma DNA of severe hepatitis patients and study its clinical application in diagnosis of severe hepatitis comparing with ALT.Methods Six milliliters of peripheral blood samples were collected from 185 patients with hepatitis B which are divided into four groups, severe hepatitis with 30 cases, acute hepatitis with 20 cases, chronic B hepatitis with 90 cases, and liver cirrhosis with 45 cases. 100 healthy controls were enrolled. Circulating DNA was extracted from plasma by the BILATEST virus DNA/RNA extraction kit and quantified with a novel duplex real-time PCR assay, respectively.Results Plasma DNA levels of hepatitis B patients were significantly higher than those of healthy controls (104.2 ng/ml vs. 23.4 ng/ml (median),P=0.0000).A significant difference of plasma DNA concentration was found between severe hepatitis and acute hepatitis (P=0.0018), and chronic B hepatitis (P=0.0000), and liver cirrhosis (P=0.0000).The median value of serum ALT of hepatitis B patients was 107.5 U/L, much higher than that of the healthy controls (24.1 U/L,P=0.0000).The levels of serum ALT were significantly different between severe hepatitis and acute hepatitis (P=0.0024), while there was no remarkable difference between severe hepatitis and chronic B hepatitis (P=0.0600), liver cirrhosis (P=1.0000). Moreover, for distinguishing severe hepatitis from liver cirrhosis and chronic B hepatitis, the plasma DNA assay was notably superior to ALT by the analysis of receive operating characteristic (ROC) curves (AUC, 0.95 vs. 0.51,P=0.0000; 0.86 vs. 0.34,P=0.0000).Conclusion The results by measuring plasma DNA of hepatitis B patients with the novel duplex real-time quantitative PCR showed that plasma DNA may be considered as a robust predictive marker for accurately evaluating hepatocyte injury degree of severe hepatitis patients.