Objective: To prepare monoclonal antibody against human thyroglobulin and establish a highly sensitive method to detect human thyroglobulin. Methods: BALB/c mice were subcutaneously immunized by native human thyroglobulin, the splenocytes of immunized mice were fused with SP2/0 myeloma cells by a routine method. The hybridoma cells secreting specific antibody were detected. The stability of the obtained hybridoma cells and the specificity of antithyroglobulin monoclonal antibody the hybridoma cells secreted were identified. Based on all above, the double antibody sandwich ELISA method was established to detect thyroglobulin. Results: Seven hybridoma cell lines which could secrete monoclonal antibody stably were obtained, and ELISA indicated the antibodies had a good specificity and sensitivity to Tg antigen. A sandwich ELISA kit for detecting human thyroglobulin was established, and its sensitivity reached to 1 ng/mL. Conclusion: The antithyroglobulin monoclonal antibodies were prepared and a sandwich ELISA kit for the detection of human thyroglobulin was established successfully, which would provide a raw material for the development of Tg rapid diagnostic kits.