A novel separation-free electrochemical immunosensor for carcinoma antigen- 125 (CA 125) was proposed based on the immobilization of CA125 antigen on colloidal gold nanoparticles that was stabilized with cellulose acetate membrane on a glassy carbon electrode. A competitive immunoassay format was employed to detect CA125 antigen with horseradish peroxidase (HRP) labeled CA125 antibody as tracer, o-phenylenediamine and hydrogen peroxide as enzyme substrates. After the immunosensor was incubated with a mixture of HRP labeled CA125 antibody and CA125 sample at 35 degrees C for 50 min, the amperometric response decreased with an increasing CA125 concentration in the sample solution. The decreased percentage of the electrocatalytic current was proportional to CA125 concentration ranging from 0 to 30 U ml(-1) with a detection limit of 1.73 U ml(-1) (S/N = 3). The proposed immunosensor showed good stability, acceptable accuracy, and would be applicable to clinical immunoassay of CA125. (C) 2005 Elsevier Ltd. All rights reserved.