The synergistic effect between graphene sheet (GS) and cobalt hexacyanoferrate nanoparticle (CoNP) was investigated, and showed that the electroactivity of CoNP was greatly enhanced in the presence of GS due to great electron-transfer ability of GS. A label-free electrochemical immunosensor for the sensitive detection of prostate specific antigen (PSA) was fabricated. Molecule 1-pyrenebutanoic acid, succinimidyl ester (PBSE) was adsorbed onto GS and the colloidal solution containing GS-CoNP-PBSE was added onto glassy carbon electrode surface to form stable thin film with high electroactivity. After anti-PSA antibody was conjugated onto PBSE, the modified electrode could be used as an amperometric immunosensor for the detection of PSA. The specific antibody-antigen immunocomplex formed on the electrode resulted in the decrease of amperometric signal of the electrode. The amperometric signal decreased linearly with PSA concentration in the range of 0.02-2 ng/mL with a low detection limit of 0.01 ng/mL. The immunosensor has the advantages of high sensitivity, good selectivity and stability, and could become a promising technique for cancer marker detection. (C) 2011 Elsevier B.V. All rights reserved.