The aim of this work was to establish a highly sensitive quantitative colloidal gold immunochromatographic assay with a test strip reader for the detection of aflatoxin B₁ using colloidal gold as label. When the labeling pH was 6.0, the concentration of antigen on T-line was 1.5 mg/mL, the concentration of antibody labeled on the colloidal gold was 4 μg/mL, and the dosage of gold-labeled antibody was 1.2 μL, the sensitivity of the assay was 5.7 ng/L. The results of accelerated aging experiment indicated that the strips shelf time was more than a year. It was proved that this method can meet the needs of the rapid detection of food when the strip was applied to detect cereals and soybean sauce.