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[Display omitted] • QD/H 2 O 2 chemiluminescence system was successfully used in HRP assay and immunoassay. • The calculated Km of HRP was more reliable compared to the colorimetric method. • A broader linear range and improved LOD value was exhibited in T4 detection. • The proposed method showed a significant correlation with the commercial ELISA. • High efficiency of the method can be applied in sensitive detection technologies. Horseradish peroxidase (HRP) is usually used as a label enzyme in immunoassay so the method used for HRP detection in enzyme immunoassay (EIA) plays a key role in sensitivity and precision. The catalytic activity of HRP does not strictly follow classic Michaelis-Menten kinetics, probably due to the inactivation of the enzyme at high concentrations of H 2 O 2. In this paper, a highly sensitive alternative procedure for the HRP assay using H 2 O 2 -sensitive CdTe quantum dots as a chemiluminescence (CL) system is reported. This method can measure a much more accurate and reliable value of K m (187 mM H 2 O 2) in comparison with the standard detection method. This system also was applied to thyroid hormone (T4) detection using HRP-based immunoassay. The QD/H 2 O 2 system exhibits a higher linear range of 0.2–16 μg/dL with the improved LOD value of 0.06 μg/dL and selective response to T4, which was better than the commercial colorimetric immunoassay. Meanwhile, the proposed method has been successfully applied to the clinical determination of T4 in the serum samples, and the results confirmed an excellent correlation with the conventional ELISA method (R2 = 0.9832), indicating the potential applications of the method for clinical diagnosis as well. [ABSTRACT FROM AUTHOR]