Object To develop an immunoassay method of species-specific protein for Radix Glycyrrhizae (RG) identification. Methods The crude drug of RG as a model was studied in this paper,the antigen of RG protein (RGP) was screened by Western-blot analysis using anti-RG serum. After species-specific protein,RGP was purified,and rabbit antiserum against RGP was prepared,the rabbit IgG of anti-RGP was isolated and biotinylated,then the sandwich enzyme-linked immuosorbent assay (ELISA) was developed. Results The method was only specific to RG,which was harvested from various areas or species,and showed excellent sensitivity and reproducibility. Conclusion The result suggests that the immunoassay method using specific antigen of RGP as a detection objective be a new potential for the unequivocal identification and quality control of RG.