Hepatitis B virus (HBV) has been always threatening to health. It is significant to establish an effective strategy for HBV carriers and hepatitis B patients diagnosis at early stage of infection in clinic. Herein, we combined nested PCR, chemiluminescence assay and magnetic separation technology to establish a highly sensitive and stable strategy for low-abundance HBV-DNA detection. Six types of monodisperse Fe₃O₄ magnetic beads (MBs) were fabricated and coated with SiO₂ layer (MBs@SiO₂). The effect of MBs@SiO₂ size on HBV-DNA extraction and chemiluminescence assay was explored, and results displayed that the MBs@SiO₂-F particles with largest size were much more suitable for HBV-DNA extraction than others, and MBs@SiO₂-C with the size range between 330 nm-450 nm performed best in chemiluminescence assay. As one of key factors, the concentration (or density) of the probe fixed on MBs@SiO₂ surface was optimized for chemiluminescence assay, exhibiting the best assay result when reaction system contained 2.0 μM of probe. Finally, under the best reaction conditions, low-abundance HBV serum specimens were sensitively and stably detected, and the result of HBV specimen with 34 copy/mL loads showed significant difference with negative and blank (p < 0.001).