In order to develop an enzyme immunoassay of glycyrrhizin (GL), N-(glycyrrhizinyl)-ε-aminohexanoic acid and -trans-4-aminomethylcyclohexanecarboxylic acid were synthesized as haptens from GL via 6', 6"-di-tert-butyl GL (VI) as a key intermediate, which was obtained by the selective tert-butylation of GL with O-tert-butyl-N, N'-dicyclohexylisourea or by the hydrogenolysis of 6', 6"-di-tert-butyl-30-benzyl GL (V) over palladium carbon. Coupling of the hapten with bovine serum albumin (BSA) (carrier protein) and β-galactosidase (labelled enzyme) was carried out by the N-hydroxysuccinimide ester method. Anti-GL serum was elicited in rabbits by immunization with N-(glycyrrhizinyl)-ε-aminohexanoic acid-BSA conjugate (XIV). Separation of bound and free fractions was performed by a double antibody method using a goat antiserum to rabbit IgG. 7-β-D-Galactopyranosyl-4-methylcoumarin was used as the substrate for the fluorometric assay of β-galactosidase activity. A satisfactory standard curve for GL was obtained in the range of 0.2-20 ng/ml.