Abstract A colloidal gold immunochromatographic strip (ICS) test based on competitive format was developed for the rapid simultaneous detection of deoxynivalenol (DON) and zearalenone (ZEN) in wheat and maize samples. For gold-based ICS test, antigen (DON-CBSA and ZEN-BSA) and goat anti-mouse IgG were respectively drawn on NC membrane as two test line (T1 and T2 line) and control line (C line), respectively. Monoclonal antibody (MAb)-gold conjugates (anti-DON MAb-gold and anti-ZEN MAb-gold) were sprayed onto the conjugate pad. To perform the test, 5 g of sample was extracted in a ratio of 1:5 with 50% methanol–water (v/v) by shaking for 3 min and the extract directly used without further cleanup steps. Matrix interference was eliminated by 2.5-fold dilutions of water extracts with buffer. The ICS test, which has cut-off levels of 100 ng/mL (1000 μg/kg) and 6 ng/mL (60 μg/kg) for DON and ZEN, respectively, and can be completed in 5 min. Analysis of DON and ZEN in wheat and maize samples revealed that data obtained from ICS test were in a good agreement with those obtained from ELISA and instrumental analysis (GC/MS for DON determination and HPLC for ZEN determination). The results demonstrate that the ICS test can be used as a reliable, rapid, cost-effective and convenient qualitative tool for on-site screening technique for the simultaneous determination of DON and ZEN in wheat and maize samples without special instrumentation.