In the present work, an indirect competitive electrochemical enzyme linked immunosorbent assay (ELISA) has been used for determination of aflatoxin B1 (AFB1) in barley. The method involves the use of disposable screen‐printed carbon electrodes (SPCEs) and anti‐aflatoxin B1 monoclonal antibodies (MAb) for immunosensor development.The specificity of the assay was assessed by studying the cross‐reactivity of the MAb relative to AFB1. The results indicated that the MAb could readily distinguish AFB1 from other toxins, with the exception of AFG1.The stability of the coating reagents was evaluated using SPCEs coated with AFB1‐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 4°C.Prior to evaluating the performance of the electrochemical immunosensor for AFB1 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. The linear range was 0.1–10 ng mL, and recoveries ranged from 100%–125%. The results obtained were confirmed by high performance liquid chromatography (HPLC) coupled with fluorescence detection. These results demonstrated the suitability of the proposed method for routine screening of AFB1 in barley.