Botulinum neurotoxins (BoNTs) are some of the most potent biological toxins. High-affinity monoclonal antibodies (mAbs) have been developed for the detection of BoNT serotypes A and B using a chemiluminescent capture enzyme-linked immunosorbent assay (ELISA). In an effort to improve toxin detection levels in complex matrices such as food and sera, we evaluated the performance of existing antitoxin mAbs using a new electrochemiluminescence (ECL) immunoassay platform developed by Meso Scale Discovery. In side-by-side comparisons, the limits of detection (LODs) observed for ELISA and the ECL immunoassay for BoNT/A were 12 and 3 pg/mL, and for BoNT/B, they were 17 and 13 pg/mL, respectively. Both the ELISA and the ECL method were more sensitive than the "gold standard" mouse bioassay. The ECL assay outperformed ELISA in detection sensitivity in most of the food matrices fortified with BoNT/A and in some foods spiked with BoNT/B. Both the ELISA and the ECL immunoassay platforms are fast, simple alternatives for use in the routine detection of BoNTs in food and animal sera.