An indirect enzyme-linked immunosorbent assay (ELISA) for aflatoxin B1 (AFB1) was developed. The method involves coating AFB1-polylysine conjugate on microtiter plates as immobilized antigen, followed by incubation with free toxin standard or sample extract and anti-aflatoxin antibody from rabbits. The amount of antibody bound to the solid phase was determined by subsequent incubation with a secondary antibody conjugated with an enzyme, i.e., goat anti-rabbit IgG-horseradish peroxidase conjugate, and reaction with the chromogenic substrate. Aflatoxins were extracted from peanut butter and corn meal samples according to the BF and CB method of the Association of Official Analytical Chemists, respectively. Extracts without column cleanup treatment were dissolved in assay buffer for subsequent ELISA. Using this technique, 79.5 to 98.6% and 68 to 97% of AFB1 added in the range of 5 to 40 (μg/kg to the corn meal and peanut butter samples were recovered, respectively. The indirect ELISA achieved the same sensitivity and specificity for AFB1 as that obtained from the direct ELISA, with an additional advantage that much less antibody (100 times less) was required for the assay.