Novel magnetic-antimicrobial-fluorescent multifunctional hybrid microspheres with well-defined nanostructure were synthesized by the aid of a poly(glycidyl methacrylate) (PGMA) template. The hybrid microspheres were fully characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared (FTIR), X-ray diffraction (XRD) and digital fluorescence microscope. The as-synthesized microspheres PGMA, amino-modified PGMA (NH2-PGMA) and magnetic PGMA (M-PGMA) have a spherical shape with a smooth surface and fine monodispersity. M-PGMA microspheres are super-paramagnetic, and their saturated magnetic field is 4.608 emu·g-1, which made M-PGMA efficiently separable from aqueous solution by an external magnetic field. After poly(haxemethylene guanidine hydrochloride) (PHGH) functionalization, the resultant microspheres exhibit excellent antibacterial performance against both Gram-positive and Gram-negative bacteria. The fluorescence feature originating from the quantum dot CdTe endowed the hybrid microspheres with biological functions, such as targeted localization and biological monitoring functions. Combination of magnetism, antibiosis and fluorescence into one single hybrid microsphere opens up the possibility of the extensive study of multifunctional materials and widens the potential applications.