Purpose: Immunoassay is susceptible to interference by other substances in the serum. The main substances interfering with thyroid function testing include heterophilic antibody, biotin, thyroid hormone autoantibody, and Macro-TSH. We reported a patient with fraudulently elevated FT4 and TSH and described various common experimental methods used to explore the existence of substances interfering with thyroid function testing.Methods and Results: FT4 and TSH were significantly lower when measured on the Architect platform (FT4: 7.09 pmol/L, TSH: 58.94 µIU/ml). Polyethylene glycol precipitation showed lower FT4 and TSH, suggesting the presence of a high molecular weight interfering substance. Heterophile blocking tube study showed heterophilic antibody interfered with TSH detection. 125I-hTSH binding study and radioimmunoprecipitation assay indicated that the patient didn’t contain anti-TSH autoantibody. The new generation of Elecsys immunoassay kit indicated that biotin interfered with TSH detection. Radioimmunoprecipitation assay showed that all four kinds of thyroid hormone autoantibodies were positive. After reviewing 24 literatures, we provided the diagnostic strategy for investigation of interferences with thyroid function immunoassays.Conclusion: We reported a case with falsely elevated TSH due to the combined action of heterophilic antibody and biotin and fraudulently elevated FT4 caused by thyroid hormone autoantibody. When there is a discrepancy between thyroid function and clinical manifestation, the presence of immunoassay interference with one or more indicators needs to be considered.