A novel magnetic polymer microsphere with amide groups and carboxyl groups was synthesized and reported here. The azidocarbonyl groups were derived from amide groups and linked to the proteins to investigate their immobilization capacity. The morphology, size, functional groups and magnetic properties of magnetic microspheres were characterized by optical microscopy, particle size analyzer, atom force microscopy, magnetic force microscopy, fourier transform infrared spectrometer, vibrating-sample magnetometer and thermal gravimetric analysis. The results indicated that the magnetic polymer microspheres had a well spherical shape with the size ranging from 1 to 10 mu m, highly re-active functional groups, superparamagnetism and strong magnetic responsibility with saturation magnetization of 18.443 emu/g and Fe3O4 content around 21%. The immobilization capacity (eta) was over 70%, The novel azidocarbonyl magnetic polymer microspheres showed potentials to be a good magnetic support and promising applications in bioseparation and biomedical fields. (C) 2009 Wiley Periodicals, Inc. J Appl Polym Sci 112: 2383-2390, 2009