Real-time polymerase chain reaction (PCR) is the current standard for serum HBV DNA measurement. However, conventional real-time PCR assays have technical limitations, and are not sensitive enough to detect low-level residual viremia in chronic hepatitis B (CHB) patients. We developed and validated a droplet digital PCR (ddPCR) assay for high-sensitivity detection of serum HBV DNA. A ddPCR assay was developed on the QX200 ddPCR System (Bio-Rad) for detection of serum HBV DNA in 200 μL of serum. The primers and probe were designed to target a highly-conserved region in the HBV X gene. The AcroMetrix HBV Panel (Thermo Fisher Scientific) and CHB patient samples were used for validation experiments to determine the assay sensitivity, specificity, linearity, intra-run variability, and inter-run variability. The ddPCR assay demonstrated lower limit of detection of 1.6 IU/mL and lower limit of quantification of 9.4 IU/mL for serum HBV DNA in probit regression. The assay also achieved excellent specificity (96.2%), linearity (R = 0.994, R