The development of novel nanomaterials brings new opportunity and challenge for high sensing detection of biomolecules. The authors describe the preparation of 3-dimentional hollow graphene balls (3D HGBs) using nickel nanoparticles (Ni-NPs) as the template. The Ni-NPs were synthesized by chemical reduction of nickel chloride and then graphene was coated onto their surface via carburization and carbonization. After etching Ni-NPs, 3D HGBs with few layers and a typical size of 100 nm were obtained. They were sprayed onto indium tin oxide glass to obtain a working electrode for electrochemical determination of levodopa in the presence of uric acid. Due to the unique hollow porous structure of the 3D HGBs, the electrode exhibits a sensitivity of 0.69 mu A.mu M-1.cm(-2) and a 1 mu M limit of detection. It is selective, reproducible and stable. It was applied to the determination of levodopa in spiked human plasma samples and it is of potential use in clinical research.