This paper reports the assembly of a disposable electrochemical immunosensor based on the indirect competitive enzyme linked immunosorbent assay (ELISA), for simple and fast measurement of aflatoxin B1 (AFB 1) in barley using differential pulse voltammetry (DPV). The immunosensor strip was assembled immobilising the biological component (i.e. the AFB1 conjugated to bovine serum albumin, incubation the sample (or standard) with the monoclonal antibody anti-AFB1 (MAb). A spectrophotometric ELISA was used in a preliminary phase of development, prior to transferring the assay to the SPEs. Results showed a detection limit of 20 and 30 pg/mL for the spectrophotometric ELISA and the electrochemical immunosensor, respectively. The extraction efficiency and the matrix effect have been evaluated by spiking blank barley with AFB1 before and after the sample treatment. After treatment, samples were analysed using a 1:10 (v/v) dilution in PBS (phosphate-buffered saline, pH 7.4) in order to minimise the matrix effect. Good recoveries were obtained, which demonstrated the suitability of the proposed method for routine screening of AFB1 in barley. © 2004 Elsevier B.V. All rights reserved.