A novel immunosensor for rapid separation-free determination of carcinoembryonic antigen (CEA) in human serum is proposed. The immunosensor is prepared by co-immobilizing thionine and horseradish peroxidase (HRP)-labeled CEA antibody on a glassy carbon electrode (GCE) through covalently binding them to GCE with a glutaraldehyde (GA) linkage. The electrochemical behavior of the immobilized thionine displays a surface-controlled electrode process with an average electron transfer rate constant of 4.74+/-2.99 s(-1). It can be used as an electron transfer mediator for enzymatic activity detection of the HRP-labeled antibody to CEA. After the immunosensor is incubated with CEA solution at 23 degrees C for 40 min, the access of activity center of the HRP to thionine is partly inhibited, which leads to a linear decrease in the catalytic efficiency of the HRP to the oxidation of immobilized thionine by H(2)O(2) at -300 mV over two CEA concentration ranges from 0.5 to 3.0 and 3.0 to 167 ng/ml. Under optimal conditions, the detection limit for the CEA immunoassay is 0.1 ng/ml at three times background noise. The immunosensor shows good accuracy and acceptable storage stability, precision and reproducibility with intra-assay CVs of 6.1% and 5.8% at 2.5 and 50 ng/ml CEA, respectively, and an inter-assay CV of 6.3% at 50 ng/ml. This method is economical and shortens the analytical time, making it potentially attractive for clinical immunoassays.