A novel electrochemical immunosensor using screen-printed carbon electrodes and functionalized magnetic particles was developed for the determination of cortisol. Anti-cortisol antibody was immobilized onto protein A-modified magnetic particles and a direct competitive immunoassay involving cortisol antigen labeled with alkaline phosphatase (AP) was employed. The resulting conjugate was trapped on the surface of the screen-printed electrode with a small magnet. Cortisol quantification was accomplished by using 1-naphthyl phosphate as AP substrate and differential pulse voltammetry for the detection of the formed 1-naphthol. The variables involved in the preparation of the immunosensor and in the electrochemical detection reaction were optimized. The calibration plot obtained for cortisol exhibited a linear range between 5.0 x 10(-3) and 150 ng mL(-1) (r = 0.993). The limit of detection was 3.5 pg mL(-1), and the EC(50) was 0.19 ng mL(-1). These results demonstrate the high sensitivity achieved with this immunosensor design which compares favourably with other previous reports. The immunosensor also exhibited a high selectivity with respect to other corticosteroid compounds closely related to cortisol. The usefulness of the immunosensor for the analysis of real samples was demonstrated by analyzing certified human sera containing cortisol at two different concentration levels.