Novel bifunctional terbium complex-based nanoparticles were developed using a modified Stöber method and a layer-by-layer assembly process. A magnetic core of Fe 3 O 4 nanoparticles was coated with a silica shell to form the first layer. Then a ternary Tb 3+ complex (TESPPA-Tb), which acted as a luminescent marker, was covalently bound to the silica surface by stable Si-O-Si bonds. The TESPPA monomer was synthesized by binding pyridine 2,6-dicarboxylic acid to 3-aminopropyltriethoxysilane, which was used as a ligand for coordination with the Tb 3+ ions. An outer shell of silica was applied to the nanoparticles to allow for versatility with surface functionalization. The nanoparticles were characterized by X-ray powder diffraction, transmission electron microscopy, Fourier transform infrared spectroscopy, ultraviolet-visible spectroscopy, vibration sample magnetometer, and photoluminescence spectroscopy. The bifunctional nanoparticles exhibited favorable superparamagnetic behavior and photoluminescence properties of Tb . Superparamagnetic NPs can be attracted by a magnetic field, but retain no residual magnetism after the field is removed, which means they can be easily separated from a matrix without agglomeration after removal of the magnetic field. Microor nanocomposites that combine superparamagnetic and optical properties into a single system have potential applications in the biomedical and biopharmaceutical fields and have attracted much attention [10]. Luminescent NPs such as quantum dots and dye-doped NPs have been used as biolabels in bioassays, and these have advantages over traditional fluorophores [11][12][13]. However, quantum dots have poor water-solubility, difficult surface conjugation chemistry, and possible toxicity in vivo, and are still under investigation [14,15]. Organic dye-doped NPs have broad emission and small Stokes shift, which results in interference between the excitation and emission signals. These limitations hinder the application of quantum dots and dye-doped NPs.Compared with these fluorophores, luminescent lanthanide complexes, especially those of Tb 3+ and Eu 3+ , have large Stokes shifts and strong narrow emission bands in the visible region, which are crucial for low detection limits and high sensitivity in fluorescence detection. They also have long fluorescence lifetimes (micro-to milliseconds range), which allow the removal of background fluorescence and increased assay sensitivity in time-resolved measurements, and can provide accurate and highly sensitive quantification