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Background: Since vascular endothelial growth factor (VEGF) is a significant regulator of cancer angiogenesis, it is essential to develop a technology for its sensitive detection. Herein, we sensitized a chemiluminescence (CL) immunoassay through the combination of H2O2-sensitive TGA-CdTe quantum dot (QD) as signal transduction, dextran as a cross-linker to prepare enzyme-labeled antigen and the ultrahigh bioactivity of catalase (CAT) as reporter enzyme. Results: Under the optimized experimental conditions, the chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA) method can detect VEGF in the excellent linear range of 2–35,000 pg mL−1, with a detection limit (S/N = 3) of 0.5 pg mL−1 which was approximately ten times lower than the commercial colorimetric immunoassay. This proposed method has been successfully applied to the clinical determination of VEGF in the human serum samples, and the results illustrated an excellent correlation with the conventional ELISA method (R2 = 0.997). The suitable recovery rate of the method in the serum ranged from 97 to 107%, with a relative standard deviation of 1.2% to 13.4%. Conclusions: The novel immunoassay proposes a highly sensitive, specific, and stable method for very low levels detection of VEGF that can be used in the primary diagnosis of tumors. With the well-designed sensing platform, this approach has a broad potential to be applied for quantitative analysis of numerous disease-related protein biomarkers for which antibodies are available. [ABSTRACT FROM AUTHOR]