Using commercially available antibodies and toxoids as templates, an ELISA, immuno-quantitative PCR (iqPCR), and multiplex immuno-PCR (iPCR) were developed for detection of Clostridium botulinum neurotoxins A and B. The obtained sensitivities for ELISA ranged from 1 ng/ml in PBS + 1% BSA to 15 and 10 ng/ml in skimmed milk for botulinum neurotoxins (BoNT)/A and BoNT/B, respectively. In semi-fat milk, the limit of detection (LOD) for both toxoids was 30 ng/ml. Quantitative immuno-PCR (iqPCR) had an LOD of 4.5-9 pg/reaction for BoNT/A in both PBS and semi-fat milk, while this was 18.5-37 pg/reaction for BoNT/B in PBS + 1% BSA and semi-fat milk, respectively. The sensitivities of ELISA and iqPCR were improved to 0.5 ng/ml and 3.75 pg/ml (0.2 pg/reaction) in semi-fat milk, respectively, when toxoid of BoNT/A was substituted with actual toxin. Multiplex iPCR with both toxoids run in the same reaction was able to distinguish presence/absence of tested BoNT/A and BoNT/B at 25 pg/reaction. The tested system offers a realistic alternative with much better sensitivity to the standard mouse assay.