Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is essential to establish and sustain viral replication. We recently reported a technique involving HBV recombinant cccDNA (rcccDNA) using Cre/loxP-mediated DNA recombination (rcccDNA^loxP). rcccDNA^loxP could be substantially induced in the nuclei of hepatocytes, which thus represents a useful surrogate for HBV cccDNA-related investigations. In the present study, we described an approach to prepare rcccDNA in an Escherichia coli (E. coli) strain ZYCY10P3S2T based on PhiC31-mediated site-specific recombination (rcccDNA^attR). Purified rcccDNA^attR minicircles were supercoils which demonstrated to support functional HBV expression and replication in the cell culture. Using the technique of hydrodynamic injection, rcccDNA^attR induced significantly prolonged HBV antigenemia in immunocompetent mice, as compared to a regular plasmid encoding linear HBV replicon. Collectively, our study provided a simplified method to surrogate HBV cccDNA in the prokaryotic expression system and in the mouse model. Our results suggested again that rcccDNA is intrinsically stable and could act as a prototype for modeling HBV persistence in mouse livers.