In the present study, a novel and efficient immobilization for horseradish peroxidase (HRP) had been developed by using 6-arm magnetic composite microsphere (Fe3O4@PAA-6-arm-PEG-NH2) containing 6-arm polyethylene glycol (6-arm-PEG-NF2) and Fe3O4. The morphology and chemical properties of Fe3O4@PAA-6-arm-PEG-NH2 were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectra (FrIR), X-ray powder diffraction (XRD), vibrating sample magnetometer (VW) and thermogravimetric analysis (TGA). The immobilization conditions and loading capacity of the carrier toward HRP were also investigated. According to the results, the optimum immobilization conditions were as follows: glutaraldehyde concentration of 0.10 mol L-1, the carrier concentration of 7 mg L-1, the temperature of 35 degrees C and immobilization time of 180 min. The loading capacity of the immobilized HRP arrived to 139.82 mg C I (RSD = 0.87%). Compared with free HRP, the stability of the immobilized HRP had been enhanced in a wide range of temperature and pH. The activity of the immobilized HRP retained 71.05% of its initial activity after 60 days storage. It was also found that the activity of the immobilized HRP retained 61.06% of its initial activity after eight times of successive reuse. The immobilized HRP could hydrolyze phenol to 94.4% within 10 min. It proved that the immobilized HRP could improve the performance of HRP, which had a good prospect in biological application.