A newly selected antibody enzyme immunoassay (SAEIA) for aqueous hoelen extract was developed. Hoelen fragments were immunized and an antiserum specific for hoelen was elicited in rabbits. An aqueous extract of hoelen was converted to a solid-phase antigen using microtiter plates. The efficiency for immobilization of hoelen extract to three types of microtiter plates was compared and carbo-plate was chosen as the solid-phase antigen support. A new assay method for a hoelen extract, with a working range between 3 ng and 30 μg, was developed applying the novel assay principle of SAEIA. The selective binding of the antibodies specific for hoelen extract, contained in the anti-hoelen serum, to the solid-phase antigen, was detected using β-D-galactosidase labeled anti-rabbit immunoglobulin G as a tracer. Specificity of the SAEIA to the hoelen extract was also demonstrated. The contents of hoelen extract in three Chinese traditional medicines were successfully analyzed applying the SAEIA method.