The invention relates to a preparation method and an application of a magnetic fluorescent nanoparticle with a shell-core structure. Firstly, a silica magnetic microsphere with a shell-core structure is prepared by using one or more than one of nanoparticles of Fe3O4, gamma-Fe2O3, MeFe2O4 (Me=Co, Mn, Ni), metal Ni, Co, Fe, and alloy Fe-Co, Ni-Fe as the inner core, and coating a silica shell, and then a fluorescent material (a chelate of Eu3+, Sm3+, Dy3+, Tb3+ and the like) is absorbed on the silica shell. Then, a layer of silica is coated on the surface to improve the stability of the fluorescent magnetic microsphere, and to prevent agglomeration and fluorescent material leakage. A lot of rare earth fluorescent materials are wrapped in the shell layer, so the fluorescence intensity signal of a prepared sample is greatly increased. The nanoparticle has dual functions of enrichment and marking, and has wider application prospects in the biomedical field.