Hepatitis B is a life-threatening liver infection caused by the hepatitis B virus (HBV). Chronic HBV infection is a major global health issue, causing an estimated 750,000 of deaths annually. Despite intense interest of biomedical and clinical researchers, the lack of an accurate, selective, and simple diagnostic approach for the early stage of the disease remains a major challenge. In this work, we developed a strand displacement amplification (SDA)-assisted G-quadruplex (GQ)-graphene oxide (GO) system for the accurate quantification of the HBV gene. Iridium (III) complex 2a, a red-emitting luminophore with a large Stokes shift and long-lived phosphorescence, was identified as the most effective probe for the split GQ-GO system among the eight selected complexes in the systematic optimization campaign. The length of nucleic acid bases in between the critical positions of the split GQ hybridization was also optimized using circular dichroism (CD) and phosphorescence experiments. Important rules on the proximity and flexibility of the stable split GQ-GO system were developed for the first time. This approach was selective for the wild HBV gene over the interfering anions, cations, proteins, and the mutated HBV genes differing by only one nucleotide base. Compared to the simple GQ-GO system, the SDA-assisted GQ-GO system produced a 211% higher phosphorescence restoration signal. The ability of the SDA-assisted GQ-GO system to detect the HBV gene in human serum and sheep red blood cells demonstrates the feasibility of using this system in practical applications.