Abstract: Luminescent nanoparticles, preparation, and application thereof are disclosed. The luminescent nanoparticle consists of matrix, which is a macromolecular compound containing carboxyl group, and a rare-earth luminescent dye dispersed in the matrix. The preparation method of the luminescent nanoparticle comprises: dissolving the rare-earth complex luminescent dye and the macromolecular compound in organic solvent miscible with water, adding the solution into water, and forming the luminescent nanoparticle by coprecipitation-selfassembly process. The prepared luminescent nanoparticle has excellent long-wave excitational luminescent properties and good stability, and can be used in coupling the surface carboxyl group of a biomolecule. The biological probes based on such luminescent nanoparticles have wide application prospects on the aspects of high-sensitivity luminescent immunoassay, biological imaging and the like.