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• An RRS assay has been developed for the detection of Eca109 cell. • Antibody and aptamer multifunctional gold nanoparticles are used as cell probe. • The sensor has been successfully applied in serum samples. Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers in China. Early and accurate diagnosis is the key to effective treatment of this disease. In this work, a resonance Rayleigh scattering (RRS) strategy was established to determine the concentration of epidermal growth factor receptor (EGFR) over-expressed ESCC cancer cells, which based on the selective binding of the anti-EGFR aptamer (Apt) and anti-EGFR antibody (Ab) multi-functionalized gold nanoparticles (AuNPs) probe (Apt-AuNPs-Ab) to the EGFR-positive cancer cell. When the probe was mixed with the Eca109 ESCC cancer cell, a distinct RRS intensity increase was observed. The probe possesses a dynamic range from 1.0 × 102-5.0 × 105 cell·mL−1 for Eca109 cells, and the detection limit is 20 cell·mL−1. Experimental results with the A2780 ovarian cancer cells line, as well as with the Eca109 ESCC cancer cell line, clearly demonstrated that the RRS assay was sensitive and selective to Eca109 than the other cancer cell lines that express a low level of EGFR. The proposed RRS assay provided a novel platform for detection of EGFR-positive cancer cells and shown the good potential for clinical diagnostics. [ABSTRACT FROM AUTHOR]