Abstract: Objective To evaluate the performance of highly sensitive HBV DNA quantitative detection and discuss its application in the treatment monitoring of hepatitis B patients with low viral load. Methods Automatic nucleic acid extraction and fluorescence PCR analysis system was used to detect HBV DNA, the linear range, precision, limit of detection (LOD), limit of quantitative detection and anti-pollution capability of the method were evaluated. HBV serological markers were detected by electrochemiluminescence immunoassay. ALT was detected by automatic biochemical analyzer. And the relationship between HBV DNA, serological markers and ALT were analyzed. Results The result showed that the linear range of high sensitivity HBV DNA quantitative detection was between 50 IU/ml and 5.0108 IU/ml. The LOD of the method reached 30 IU/ml. The precision of inter and intra-run CV were all less than 5%. And the method had a strong anti-pollution capability. The patients with HBV DNA load 500 IU/ml accounted for 46.4%. In HBeAg positive or negative group, the ALT abnormal rate of patients with HBV DNA load ranging from 30 IU/ml to 500 IU/ml was significantly higher than those with HBV DNA load 30 IU/ml (P=0.012,P=0.001), but different HBV DNA load had no relationship with serological markers patterns (P=0.179). Conclusion Automatic nucleic acid extraction and fluorescence PCR analysis system based highly sensitive HBV DNA quantitative detection has good performance, which is suitable for the treatment monitoring of patients with low hepatitis B viral load. And the treatment monitoring of such hepatitis B patients should be enhanced.