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A sensitive electrochemiluminescence (ECL) biosensor for the specific DNA sequence of hepatitis C virus (HCV) was developed based on the efficient quenching effect of the ferrocene cluster functionalized gold nanoparticles (Fc@AuNPs) on the ECL of electrodeposited silica@Ru(bpy)32+‐chitosan‐graphene oxide nanocomposite (SiO2@Ru−CS−GO). Graphene oxide (GO) can accelerate electron transfer rate, thus improving the ECL of Ru(bpy)32+ on electrode surface. The molecular beacons (MB) was fixed to SiO2@Ru−CS−GO by glutaraldehyde (GA) using the Schiff reaction between amino groups of chitosan (CS) and MB. The ECL of SiO2@Ru−CS−GO was depressed greatly by the Fc@AuNPs labelled at the end of MB, then, a stronger ECL was observed when the distance between Fc@AuNPs and SiO2@Ru−CS−GO increased after the hybridization of target DNA with MB. Under optimum conditions, the restored ECL intensity increased linearly with the target DNA concentration in the range of 1.0×10−16∼1.0×10−10 mol ⋅ L−1, and the limit of detection (LOD) is 1.4×10−17 mol ⋅ L−1. The proposed method exhibits acceptable stability and reproducibility. In general, the constructed HCV biosensor can be used for the sensitive detection of HCV in human serum, suggesting potential application prospects in bioanalysis. [ABSTRACT FROM AUTHOR]