A novel amperometric immunosensor with amplified sensitivity for the determination of carcinoembryonic antigen (CEA) has been developed based on the assembly of {Thi(+)/nano-u}(n) layer-by-layer films by alternate adsorption of negatively charged gold nanoparticles (nano-Au) and positively charged thionine (Thi(+)) on Titania nanoparticles/gold nanoparticles composite film formed previously on the electrode via self-assembly and deposition method, which provided an interface to assemble carcinoembryonic antibody (anti-CEA). Subsequently, HRP was backing-filled into the CEA-modified electrode surface to amplify the response of the antigen-antibody interactions. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to monitor and confirm the films growth. Assay conditions were optimized including the incubation temperature and time, the pH of working buffer, the concentration of the H2O2 and the electrodeposition time of nano-Au, etc. The reduction current of the immunosensor decreases linearly in the range of CEA from 0.2 to 80.0 ng/mL with a detection limit of 0.07 ng/mL in presence of 0.55 mM H2O2 in working solution. Moreover, the immunosensor showed acceptable reproducibility, high sensitivity and long-term stability. Clinical serum samples were assayed with this method and the results were in acceptable agreement with those obtained from the enzyme-linked immunosorbent assays (ELISAs). Therefore, the platform that combines the advantages of nanostructured materials with those of the layer-by-layer self-assembling technique opens the doors to the new and exciting possibilities for the development of immunosensor using different transduction modes. (C) 2007 Elsevier B.V. All rights reserved.