Graves' disease (GD) is an autoimmune disease of the thyroid gland caused by autoantibodies against thyroid-stimulating hormone receptor (TSHR). Currently, the diagnostic test for TSHR autoantibodies is based on an indirect competitive binding assay that measures the ability of TSHR autoantibodies to inhibit the binding of thyroid-stimulating hormone (TSH) to TSHR. Here, we have developed a specific and direct diagnostic method for autoantibodies in GD that incorporates immobilized TSHR-containing recombinant proteoliposomes into an enzyme-linked immunosorbent assay (ELISA). To reduce non-specific binding of autoantibodies to recombinant proteoliposomes, we investigated the effect of polyethylene glycol (PEG)-lipid on the binding of commercially available anti-TSHR antibodies (aTSHRAb). The incorporation of PEG-lipids into liposomes decreased non-specific binding, as compared to liposomes that did not contain PEG-lipids, and the addition of blocking reagents further decreased non-specific reactivity. aTSHRAb exhibited higher reactivity towards PEG-modified TSHR recombinant proteoliposomes than PEG-modified liposomes without TSHR (bare liposomes). Importantly, serum autoantibodies from patients with GD, which is associated with hyperthyroidism, exhibited remarkably specific binding to TSHR recombinant proteoliposomes. Serum autoantibodies from patients with Hashimoto's disease (HD), which is associated with hypothyroidism, also reacted specifically with proteoliposomal TSHR. These results suggest that immobilized TSHR recombinant proteoliposomes can serve as a direct diagnostic test for GD and HD. Furthermore, given that there is no competition test currently available for detecting autoantibodies in HD, the combination of TSHR recombinant proteoliposome ELISA and indirect competitive TSHR binding assay might be an effective way to discriminate between GD and HD.