Licorice is utilized in various food industries around the world for seasoning agents, confectioneries, drinks, and functional foods. Glycyrrhizin (GL) and liquiritin (Liq) are major quality control chemical markers of licorice that have multifunctional bioactivities. Chemical quality control of licorice is important because its component profiles change depending environmental factors (climate, soil condition, and water deficit) and differences between species. Double eastern blotting using anti-GL and anti-Liq monoclonal antibodies was developed for more convenient, rapid, and specific quality control analysis of GL and Liq, respectively. Moreover, double eastern blotting was applied to investigate the immunohistochemical distributions of GL and Liq in the root of fresh licorice; the localization of both components was then clarified visually. This double eastern blotting technique for GL and Liq may serve as a powerful approach for visually determining the chemical quality of licorice.