Liquiritin was reacted with a keyhole limpet hemocyanin (KLH) to synthesize a liquiritin-KLH conjugate as an immunogen for mice. A hybridoma cell line named 2F8 secreted a monoclonal antibody (mAb) against liquiritin, which was applied to an enzyme-linked immunosorbent assay (ELISA) for liquiritin. ELISA showed a good linear range from 0.39 to 25 μg/mL of liquiritin. The maximum relative standard deviation (RSD) values for the intra-assay and interassay were approximately 5%. The recovery rates of liquiritin were in the range of 100.9-103.7%, and the concentrations of liquiritin in various licorice roots, as determined by ELISA, showed a good correlation with those analyzed by high-performance liquid chromatography (HPLC; R² = 0.948). These results suggested that ELISA with anti-liquiritin mAb could be a simple, rapid, convenient, and accurate method for the high-throughput analysis of liquiritin in various licorice products including liqueurs, sweets, and food supplements.