Ester-type Aconitum alkaloids (AAs), the main medicinal ingredients of Aconitum L. herbs, could cause brain and heart damage in humans and animals and have raised concerns worldwide. In the present study, we aimed to produce a high-performance and broad-spectrum antibody and establish an immunoassay method of ester-type AAs, 3-succinyl aconitine (ACO-HS) was selected as an optimal hapten from five designed haptens comparing the similarity of stereo structure, electronic distribution, and physicochemical properties using the computer-aided molecular modeling technology. The monoclonal antibody (mAb) 1A9 exhibited broad-spectrum recognition specificity of 15 ester-type AAs was obtained and had a high sensitivity with the binding affinity (half-maximum inhibition concentration, IC(50)) of 0.73-130.36 mug L(-1). Through molecular docking, it was found that mAb 1A9 and ester-type AAs showed a semi-enveloped structure through hydrogen bonds and hydrophobicity interaction. The amino acid residues that responsible for recognition were ARG107, GLU55, PRO113, VAL36, and SER64, and the critical structures to be recognized of AAs were acetyl group, benzoyl group, and N-linked carbon chains. The developed indirect competitive enzyme-linked immunosorbent assay (icELISA) based on mAb 1A9 allowed a sensitive determination of 15 ester-type AAs with the limit of detection (LOD) of 0.21-43.72 mug L(-1), and it was suitable for the analysis of ester-type AAs in various Aconitum L. samples. These results provided an effective strategy for the preparation of targeted broad-spectrum antibodies of small molecules and proposed an icELISA method available for rapid, sensitive, and high-throughput detection of toxic ester-type AAs in Aconitum L. herbs. CI - Copyright (c) 2022 Elsevier B.V. All rights reserved.