A new detection format for multiplexed analysis based on the use of magnetic fluorescent composite nanoparticles was presented in this paper. Two different antigens, Newcastle disease virus (NDV) antigen and Avian virus arthritis virus (AVAV) antigen, were conjugated to two kinds of magnetic fluorescent composite nanoparticles of different luminescent colors, while red-emitting CdTe QDs were attached to the antibody of NDV and AVAV. Both CdTe QDs-labeled antibodies and magnetic fluorescent composite nanoparticles labeled antigens were used to form a typical immunoreaction system for the detection of NDV and AVAV. Also a typical mixed separation format was realized, which showed the outstanding magnetic properties of composite nanoparticles and the broad application in immunoseparation.