Multi-functional nanoparticles possessing magnetic, up-conversion fluorescence and bio-affinity properties were synthesized and characterized. The particles have a core/shell structure. Iron oxide nanoparticles of 5–15 nm diameter were synthesized as the magnetic core. The core was covered with ytterbium and erbium co-doped sodium yttrium fluoride (NaYF4:Yb,Er), an efficient infrared-to-visible up-conversion phosphor. The phosphor shell was prepared by co-precipitation of the rare-earth metal salts with fluoride in the presence of EDTA and the magnetic nanoparticle. After the magnetic/fluorescent hybrid particle was coated with SiO2 and activated with glutaraldehyde, streptavidin was immobilized on the particle. The magnetic/fluorescent nanoparticles were found by transmission electron microscopy to be well-dispersed spherical particles with an average diameter of 68 nm. Both energy dispersive X-ray microanalysis and X-ray fluorescence spectra revealed the existence of iron in the particle. Measurements performed on a vibrating sample magnetometer obtained a strong magnetic response for the particle and fluorescence measurements demonstrated its up-conversion property. X-Ray diffraction analysis suggests the phosphor shell has the same structure as the pure NaYF4:Yb,Er nanoparticles we prepared in a previous study (G. S. Yi, H. C. Lu, S. Y. Zhao, Y. Ge, W. J. Yang, L. H. Guo, D. P. Chen and J. Cheng, submitted). Streptavidin-coated magnetic/fluorescent particles were found to bind specifically to a glass slide spotted with biotinylated IgG and emit up-conversion fluorescence, confirming the successful coating of the protein and retention of its optical activity and bio-affinity.