Aflatoxin M1 (AFM1) in milk has two standard limits, namely, the European standard set up at 0.05 ng/mL and the other one (including America, China, and other countries) at 0.5 ng/mL. A one-step immunochromatographic assay (ICA) with two cutoff values was developed to satisfy these two standard limits, wherein test zones including test 1 and 2 (T1 and T2) lines were formed by dispensing AFM1-BSA antigen onto a nitrocellulose membrane. The migrating AFM1 analyte in the sample and capture reagent immobilized on two test lines competed for the limited binding sites of AFM1 antibody, which conjugated with colloidal gold. Three red lines appeared at T1 and T2 lines and control (C) line when the sample was negative or the AFM1 analyte concentration was lower 0.05 ng/mL. However, only two red lines appeared at T1 and C lines when the AFM1 analyte concentration was 0.05 ng/mL or higher but lower 0.5 ng/mL. One red line appeared at C line when AFM1 analyte concentration was 0.5 ng/mL or higher. Results showed that the novel immunochromatographic assay was superior to traditional ICAs because of satisfying two standard limits at one step.