In the present study, a novel monoclonal antibody (MAb) specific for icariin (ICA) was prepared and characterized. A hybridoma‑secreting MAb against icariin was produced by fusing splenocytes immunized with an ICA‑bovine serum albumin conjugate with a hypoxanthine‑aminopterin‑thymidine‑sensitive mouse myeloma SP2/0 cell line. The antibody showed high specificity for ICA with almost no cross‑reactivity against the majority of structurally‑related chemicals. Subsequently, an indirect competitive enzyme‑linked immunosorbent assay (ELISA) for ICA was established and characterized. In this assay, an effective measuring range of 10‑1,000 ng/ml of ICA (R2=0.9828) was detected. Intra‑ and inter‑assay repeatability and precision were achieved with a relative standard deviation (RSD) of <10%. A mean recovery of 95‑115% was obtained, with an RSD of <10%. In addition, the levels of ICA in traditional Chinese herbal prescriptions were determined, and correlation between the ELISA and high‑performance liquid chromatography analyses of total ICA was obtained. These results demonstrated that a reliable ELISA method had been successfully developed to determine ICA in traditional Chinese herbs and may contribute to further clinical investigations.