A novel multilayer film based on gold nanoparticles (AuNPs), thionine (Thi), and TiO2–graphene (TiO2–Gr) was exploited to develop a highly sensitive amperometric immunosensor for detecting carcinoembryonic antigen (CEA). Firstly, Nafion–TiO2–Gr homogeneous composite was dropped on the surface of a glassy carbon electrode (GCE). Then Thi was chemisorbed by the TiO2–Gr–Nafion composite. Furthermore, the negative ly charged AuNPs were chemisorbed onto Thi film through the electrostatic force with the amino groups of Thi. Cyclic voltammetry (CV) was employed to characterize the assembly process and the performance of the immunosensor. Because of the synergistic effect of the AuNPs, Thi, and the unique properties of TiO2–Gr, the obtained immunosensor exhibited a wide linear response to CEA in two ranges from 0.1 to 10.0 ng mL−1 and from 10.0 to 120.0 ng mL−1 with a relatively low detection limit of 0.01 ng mL−1 (S/N = 3), as well as good stability and repeatability.
