In this study, a novel tracer, horseradish peroxidase (HRP) functionalized nanocomposites, was designed to label the signal antibodies for the detection of carcinoembryonic antigen (CEA) using an electrochemical immunosensor. To construct high-performance immunosensor, gold nanoparticles (AuNPs) dotted thionine functionalized carbon nanotubes (Thi-CNTs), which could increase the surface area, was used to immobilize the primary antibody (Abi). The preparation of the HRP functionalized nanocomposites and labeling of signal antibody were performed by one pot assembly of HRP and signal antibody on AuNPs onto polyaniline (PAN) functionalized carbon nanotubes (AuNPs-PAN@CNTs). The high content HRP on the surface of the AuNPs-PAN@CNTs amplified the detectable signal for sandwich-type immunoassay. Electrochemical impedance spectra and cyclic voltammetry are used to investigate the assemble process of the immunosensor. Furthermore, differential pulse voltammetry was utilized to monitor the response signal of immunosensor. Under the optimal conditions, the immunosensor displayed an excellent linear response for the detection of CEA ranging from 0.02 to 80.0 ng mL(-1) with a detection limit of 0.008 ng mL-1 (at 3 a.). In addition, the immuosensor was applied to serum samples, and the results obtained were agreement with the reference values. (C) 2014 Elsevier Ltd. All rights reserved.