We proposed a one-step hydrothermal procedure to form Fe3O4 nanospheres on chemically reduced graphene oxide (CRGO) surfaces, and used this nanocomposites as substrates for enzyme immobilization.The as-prepared Fe3O4/CRGO nanocomposites were characterized using scanning electron microscopy (SEM), X-ray powder diffraction (XRD), FT-IR and vibrating sample magnetometer.Fe3O4 microspheres are randomly distributed on graphene sheets, the average diameter of Fe3O4 microspheres is about 260 nm.We used Horseradish peroxidase (HRP) as a model enzyme to investigate the immobilization activity.The HRP loading was 23.3mg/g supports and retained 70% of the first use after ten cycles.The catalyzed capability of immobilized HRP was investigated and the immobilized HRP exhibited broader pH stability and excellent reusability.The results show that the Fe3O4/CRGO nanocomposites are appropriate for the immobilization of enzyme, and could have potential use in practical.