Molecular detection of HBV has a significant impact on prognosis and therapy of the disease. In this paper, a sensitive nucleic acid detection method of HBV was established taking advantage of magnetic nanoparticles (MNPs), chemiluminescence (CL) and polymerase chain reaction (PCR). HBV-DNA was extracted from hepatitis B positive human blood samples using MNPs adsorption method and biotin was labeled on the DNA segment after base insertion of bintin-dUTP in PCR. The biotinylated DNA segment was captured by amino probe immobilized on carboxyl MNPs and was detected by the chemiluminescence system of alkaline phosphatase catalyzing 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1, 2-dioxetane. Different concentrations of HBV-DNA were detected under the optimized experiment conditions and the relevant CL intensity were obtained, which provided a novel research or clinic diagnosis method for the quantification detection of HBV-DNA.