Objective To explore the feasibility of low-load hepatitis B virus (HBV) DNA S-gene amplification and optimize the experimental conditions, so as to provide a basis for the detection of HBV DNA S-gene mutation in occult HBV infection (OBI) patients. Methods HBV DNA S-gene in serum samples of 6 cases with low-load HBV DNA (100-200 IU/mL) and 22 cases with lower-load HBV DNA (20-99 IU/mL) was amplified by traditional nested polymerase chain reaction (PCR) and self-built 2-round PCR. The experimental conditions, such as primer sequences, primer amount, the dilution ratio of PCR product templates, annealing temperature, the number of PCR reaction cycles, and PCR reaction system, were optimized. The PCR amplification product was observed by agarose gel electrophoresis, the target band gel was cut for cloning and sequencing, and then the cloned sequencing result was confirmed by BLAST comparison of nucleic acid sequence. Results Three pairs of primers (P1-P3) for nested PCR were designed, and the amplified products theoretically contained the whole HBV DNA S-gene. After optimization of PCR amplification conditions, only 2 of the 6 serum samples with low-load HBV DNA were amplified by nested PCR, and 22 samples with low-load HBV DNA were failed to amplify any fragments. Twelve pairs of primers (P4-P15) were designed for the self-built 2-round PCR, and the amplified products theoretically contained the whole HBV DNA S-gene. After PCR amplification conditions were optimized and P13 was selected as the best primer, all 6 serum samples with low-load HBV DNA and 15 (15/22, 68.18%) samples with lower-load HBV DNA were amplified successfully. The PCR products were confirmed as the HBV DNA S-gene specific target sequences by cloning and sequencing. Among the 15 samples, the minimum HBV DNA load was 20.1 IU/mL. Conclusion The self-built 2-round PCR based on the primer P13 is more suitable for the amplification of low-load HBV DNA S-gene. The amplification efficiency and specificity are much better than traditional nested PCR. The amplified products can be further applied to analyze HBV DNA S-gene mutation in OBI patients.