The invention discloses a construction method of HBV (Hepatitis B Virus) isolating strain replicating-form plasmid, comprising the following steps of: 1, extraction of HBV full-length genome, PCR (Polymerase Chain Reaction) amplification and clone construction; 2, construction of a recombinant transition vector; 3, enzyme cutting and dephosphorylation of the recombinant transition vector; and 4, construction of replicating-form plasmid, wherein the recombinant transition vector is synchronously and directionally connected by three connection segments, two reverse BspQI enzyme cutting sites are formed between two inserted HBV segments, and the clone of the HBV full-length genome and the bacterial colony cultivation and amplification of the replicating-form plasmid are carried out by an SOC culture medium. The invention applies a novel full-length genome clone vector, improves the enzyme cutting solution and adopts a novel bacterial colony culture medium. The invention has the advantages of simple performance, high efficiency and stability, can be applied to the comprehensive phenotype research on a China HBV clinic isolating strain whole genome and has very important application value.