Objective To set up the rolling circle amplification (RCA) system for detecting hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), and to evaluate the specificity and sensitivity of this system. Methods Plasmids containing full-length of wild-type HBV genome were treated with restriction enzyme and T4 DNA ligase, and then were concentrated. The DNA fragments were recovered by the nucleic acid purification kit and severed as standard HBV cccDNA. Total DNA was extracted from hepatic tissues of seven chronic hepatitis B patients. RCA method was used to amplify genomes from tissue samples. Standard HBV cccDNA, 3.2 kb liner HBV DNA, normal hepatic tissue samples and 15 serum samples of patients with chronic HBV infection were used as controls to determine the specificity of RCA. Ten-fold serial dilutions of standard HBV cccDNA were used for determining the sensitivity. Results The standard HBV cccDNA was successfully constructed and could be detected by RCA method. HBV cccDNA could be amplified from 2 mg hepatic tissue samples at least of HBV infected patients, and could be detected as low as 1 ×102 copy/μL. cccDNA was not detected in 3.2 kb liner HBV DNA, normal hepatic tissue samples and 15 serum samples of chronic HBV infected patients. Conclusion RCA method can be used for rapid and simple detection of HBV cccDNA with high specificity and sensitivity.