In this work, the reduced graphene oxide-Fe3O4 nanocomposite (rGO/Fe3O4) was prepared by a facile in-situ deposition of Fe3O4 nanoparticles on graphene sheets. The fact that graphene oxide (GO) was completely reduced to graphene by Fe2+ was evidenced by X-ray diffractometer, ultraviolet visible spectrophotometer, Fourier transform infrared spectrometer and X-ray photoelectron spectroscopy. The results showed Fe3O4 nanoparticles were generated uniformly and bonded on the surface of rGO with a particle size of about 50 nm. In addition, the rGO/Fe3O4 nanocomposite showed a good conductivity and was considered as a high volume surface ratio matrix for the immobilization of glucose oxidase (GOx) on a glassy carbon electrode (GCE). The fabricated rGO/Fe3O4/GOx/GCE exhibited a fast electron transfer and good electrocatalytic activity due to the good electrochemical properties of the rGO/Fe3O4 nanocomposite. These results demonstrated that the rGO/Fe3O4 nanocomposite prepared by in-situ deposition could be applied to high performance biosensor for glucose detection.