Abstract Antibodies specific to aflatoxin B1 were generated from rabbits immunized with AFB1–bovine serum albumin (BSA). By using these antibodies, this work presents a rapid and sensitive competitive direct enzyme-linked immunosorbent assay (cdELISA) and a gold nanoparticle immunochromatographic strip method for detecting AFB1 in food and feed samples. In the rapid cdELISA, AFB1 at a concentration of 0.15 ng/ml causes 50% inhibition (IC50) of binding AFB1–horseradish peroxidase to the antibodies. Effective on-site detection capability of AFB1 is also developed based on a rapid and sensitive antibody–gold nanoparticle immunochromatographic strip method. This strip has a detection limit of 2.0 ng/ml for AFB1 in food and feed samples. Additionally, the entire analysis is completed within 10 min. Closely examining 36 food and feed samples by cdELISA reveals that 20 are contaminated with AFB1 from 1.3 to 234 ng/g. Results of 20 contaminated samples further analyzed with immunochromatographic strip assay correlate well with those obtained from cdELISA. The proposed cdELISA and immunochromatographic strip methods are highly sensitive to the rapid screening of AFB1 in food and feed samples.