A new signal amplification strategy employing nanocomposites composed of gold nanoparticles (GNPs) with DNA and methylene blue (MB) as labels has been developed to construct a novel immunosensor for the highly sensitive bioanalysis of carcinoembryonic antigen (CEA). To fabricate the label, DNA was first linked to GNPs through the formation of an Au-S bond. Next, the resulting bioconjugates were sequentially bound with MB via the guanine (G) bases in DNA specifically and then with secondary CEA antibody (Ab(2)) via the crosslinking of glutaraldehyde (GA). The receptor-analyte complex is formed by the biorecognition of the as-obtained MB labeled Ab(2) with the primary CEA antibody (Ab(1)) immobilized on the surface of a modified GNP/Chitosan (Chits) composite electrode. After the receptor-analyte complex has formed, square wave voltammetry (SWV) was employed to oxidize the labeled MB in order to detect the CEA. This immunosensor shows a linear response from 0.10 to 2.0 pg mL(-1) CEA with an improved detection limit of 0.05 pg mL(-1) as compared to the conventional immunoassay. In addition, this new protocol shows acceptable stability, reproducibility and good recovery (97.5-110.0%) for CEA in human serum with great potential for clinical applications. (C) 2011 Elsevier Ltd. All rights reserved.