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In the present work, an indirect competitive electrochemical enzyme linked immunosorbent assay (ELISA) has been used for determination of aflatoxin B 1 (AFB 1 ) in barley. The method involves the use of disposable screen-printed carbon electrodes (SPCEs) and anti-aflatoxin B 1 monoclonal antibodies (MAb) for immunosensor development.The specificity of the assay was assessed by studying the cross-reactivity of the MAb relative to AFB 1 . The results indicated that the MAb could readily distinguish AFB 1 from other toxins, with the exception of AFG 1 .The stability of the coating reagents was evaluated using SPCEs coated with AFB 1 -bovine serum albumin (BSA) conjugate. The results showed that the coated electrodes could be used for up to one month after their preparation and storage at 48C.Prior to evaluating the performance of the electrochemical immunosensor for AFB 1 with spiked samples, the effect of barley extract on assay performance was tested. Using this calibration method, the limit of detection (LOD) was found to be 90 pg mL 21 .