The colloidal gold immunochromatographic strip was developed to detect aflatoxin B₁ (AFB₁), zearalenone (ZEN) and deoxynivalenol (DON) in grains. Methods The pH of colloidal gold solution and the dosage of the specific antibody and antigen were optimized through three combinations. The limit of quantitation, accuracy, specificity and the stability of the detection strip were investigated. Results The optimal pH of colloidal gold solution for AFB₁, ZEN and DON was 7.5,7.5 and 7.0, respectively. The optimal concentration of antibody marking was 4.2,7.2 and 9.6 μg/ml, respectively. The optimal concentration of the specific antigen was 1.0,1.4 and 0.8 mg/ml, respectively. Sixty samples were detected through instrument method and colloidal gold immunochromatographic method simultaneously, and the results were consistent with each other. No cross-reaction was observed with other analogue and mycotoxins, and the triple strip could be stored at room temperature for 12 months. Conclusion The triple strip was a simple, accurate and stable method for monitoring AFB₁, ZEN and DON in grains, and had good application foreground in field analysis.