The enzyme-immobilized fluorescent magnetic iron oxide nanoparticles were prepared and characterized for the detection of glucose and urea in biological media. The magnetic iron oxide nanoparticles (MIONPs) had an average size of 30 nm in diameter. Two fluorescence dyes (ruthenium complex and aminofluorescein) were coated onto the surface of the MIONPs using silica and melamine formaldehyde (MF) polymer. The immobilization of the enzymes (GOD and urease) onto the fluorescent MIONPs was carried out by using glutaraldehyde as a cross linking reagent. The MFcoated fluorescent MIONPs had stronger fluorescent intensity and higher amounts of immobilized enzymes than the silicacoated fluorescent MIONPs. The GOD-immobilized fluorescent MIONPs could be used as bio-probes for the detection of glucose, while the urease-immobilized fluorescent MIONPs were applied to detect urea in biological processes and medical diagnostics.