Hepatitis B virus is a kind of DNA virus which can cause serious epidemic disease. The analysis and detection of sequence-specific DNA have great significance in forensic analysis, early-stage identification and treatment of genetic disorders. Chemiluminescent detection of DNA has been applied in many fields, such as biological technology and molecular biology, due to its simple operation and high sensitivity. On the other hand, owing to possessing easy magnetic separation and large surface properties, magnetic nanoparticles have also been employed as special carriers to immobilize biomolecules. In this paper, the magnetic nanoparticles are prepared by soft-template method with uniform shape and good dispersion. Then a detection method of hepatitis B virus DNA is established taking advantages of both chemilumiescence with the system of alkaline phosphatase catalyzing 3-(2 -spiroadamantane)-4-methoxy -4-(3 -phosphoryloxy) phenyl-1, 2-dioxetane and magnetic nanoparticles. The optimization of conditions affecting the hybridization reaction and the chemilumiescence detection are also investigated to promise a high sensitivity.