Three-dimensional (3D) structured graphene is a material of great interest due to its diverse applications in electronics, catalytic electrodes, and sensors. However, the preparation of 3D structured graphene is still challenging. Here, we report the fabrication of multilayer graphene balls (GBs) by template-directed carbon segregation using nickel nanoparticles (Ni-NPs) as template materials. To maintain the ball shape of the template Ni-NPs, we used a carburization process using polyol solution as the carbon source and a thermal annealing process to synthesize graphene layers via carbon segregation on the outer surface of the Ni-NPs. The resulting GBs were hollow structures composed of multilayer graphene after the removal of core Ni-NPs, and the thickness of the graphene layers and the size of GBs were tunable by controlling the graphene synthesis conditions. X-ray diffraction analysis and in situ transmission electron microscope characterization revealed that carbon atoms diffused effectively into the Ni-NPs during the carburization step, and that the diffused carbon atoms in Ni-NPs segregated and successfully formed a graphene layer on the surface of the Ni-NPs during thermal annealing. We also performed further heat treatment at high temperature to improve the quality of the graphene layer, resulting in highly crystalline GBs. The unique hollow GBs synthesized here will be useful as excellent high-rate electrode materials for electrochemical lithium storage devices.