A “simultaneous” enzyme-linked immunoassay for the measurement of anti-thyroglobulin autoantibody in human serum was studied, in which human thyroglobulin-coated silicone rubber rod, ß-D-galactosidase- or horseradish peroxidase-conjugated human thyroglobulin and serum sample were mixed at the same time. In order to obtain a suitable range of the measurable antibody, an appropriate amount of human thyroglobulin-enzyme conjugate was carefully selected. A successful simultaneous assay for antithyroglobulin was obtained using thyroglobulin-ß-D-galactosidase conjugate, in which the minimum amount of detectable antibody was approximately 10 ng/ml using 5 μl of serum. This sensitivity was 20-fold higher than that in two-step sandwich enzyme-linked immunoassay reported previously.