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Background We aimed to develop a quantitative assay to measure duck HBV (DHBV) DNA in single hepatocyte nuclei from DHBV-infected animals and to observe intranuclear DHBV DNA kinetics undergoing entecavir (ETV) therapy. Methods DHBV DNA in isolated nuclei was amplified by quantitative real-time PCR. Liver tissues from chronically-infected ducks with or without ETV treatment were assessed. Cell cycle phases were defined with flow cytometry in single nuclei. Results We successfully established a quantitative assay to measure intranuclear DHBV DNA in single nuclei with high specificity, sensitivity and acceptable interassay variations. The intranuclear viral DNA copy numbers varied dramatically (2–204 copies/nuclei) in 11 ducks with active viral replication. Average intranuclear DHBV DNA copies from individual animals (7.57–57.67 copies/nuclei) significantly correlated with total intranuclear ( r s=0.955, P s=0.745, P=0.008) viral DNA levels. The median intranuclear DHBV DNA copies in virus-positive nuclei were greater in gap 0/1 than those in gap 2/ mitosis and synthesis phases ( PConclusions Intranuclear DHBV DNA levels varied significantly, which could be partially attributed to effects of cell cycle phases, and could be decreased by ETV therapy.