A novel magnetic fluorescent encoded nanoimmunoassay system for multicomponents detection and separation of the subtrace pathogenic DNA (hepatitis B virus surface gene HBV; hepatitis A virus polyprotein gene HAV) was established based on new type of magnetic fluorescent encoded nanoparticles and sandwich immunoassay principle. Thismethod combinesmultifunctional nanoparticles, immunoassay technique, fluorescence labeling, and magnetic separation of multicomponents technology. It has many advantages such as high sensitivity, low detection limit, being easy to operate, and great potential for development.The results of this work show that, based on nanoimmunoassay system, it could quantitatively detect the multicomponents trace pathogenic HAV and HBV DNA and detection limit up to 0.1pM and 0.12 pM. Furthermore, with the improvement of the performances of magnetic fluorescent encoded nanoparticles, the sensitivity will be further improved. In this experiment, a new nanoimmunoassay system based on magnetic fluorescent encoded nanoparticles was established, which will provide a new way for the immunoassay and separation of multicomponent biomolecules.