A new kind of magnetic/luminescent multifunctional nanoparticles was synthesized by covalently linking multiple carboxyl-functionalized superparamagnetic Fe₃O₄ nanoparticles and individual amino-functionalized silica-coated fluorescent NaYF₄ : Yb,Er up-conversion nanoparticles (UCNPs). The resultant nanocomposites bear active carboxylic and amino groups on the surface that were proved to be chemically active and useful for further facile bioconjugation with biomolecules. The UCNPs in the nanocomposite particles can emit visible light in response to the irradiation by near infrared (NIR) light, enabling the application of the nanocomposites in bioimaging. X-Ray diffraction, infrared spectroscopy, transmission electron microscopy, luminescence spectroscopy, and magnetometry were applied to characterize the multifunctional nanocomposites. The nanocomposites exhibited good superparamagnetic and excellent green up-conversion photoluminescent properties that can be exploited in magnetic separation and guiding as well as bioimaging. Due to the presence of active functional groups on the nanocomposite surface, the Fe₃O₄/NaYF₄ : Yb,Er magnetic/luminescent nanocomposites were successfully conjugated with a protein called transferrin, which specifically recognizes the transferrin receptors overexpressed on HeLa cells, and can be employed for biolabeling and fluorescent imaging of HeLa cells. Because NIR light can penetrate biological samples with good depth without damaging them and can avoid autofluorescence from them, the presence of both NIR-responsive UCNPs and superparamagnetic nanoparticles in the nanocomposite particles will enable the practical application of the nanocomposites in bioimaging and separation.