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Glypican-3 (GPC3), as an emerging biomarker, has been shown to be beneficial for the early diagnosis and treatment of hepatocellular carcinoma (HCC). In this study, an ultrasensitive electrochemical biosensor for GPC3 detection has been constructed based on the hemin-reduced graphene oxide-palladium nanoparticles (H-rGO-Pd NPs) nanozyme-enhanced silver deposition signal amplification strategy. When GPC3 specifically interacted with GPC3 antibody (GPC3_Ab) and GPC3 aptamer (GPC3_Apt), an “H-rGO-Pd NPs-GPC3_Apt/GPC3/GPC3_Ab” sandwich complex was formed with peroxidase-like properties which enhanced H₂O₂ to reduce the silver (Ag) ions in solution to metallic Ag, resulting in the deposition of silver nanoparticles (Ag NPs) on the surface of the biosensor. The amount of deposited Ag, which was derived from the amount of GPC3, was quantified by the differential pulse voltammetry (DPV) method. Under ideal circumstances, the response value was linearly correlated with GPC3 concentration at 10.0–100.0 μg/mL with R² of 0.9715. When the GPC3 concentration was in the range from 0.01 to 10.0 μg/mL, the response value was logarithmically linear with the GPC3 concentration with R² of 0.9941. The limit of detection was 3.30 ng/mL at a signal-to-noise ratio of three and the sensitivity was 1.535 μAμM⁻¹cm⁻². Furthermore, the electrochemical biosensor detected the GPC3 level in actual serum samples with good recoveries (103.78–106.52%) and satisfactory relative standard deviations (RSDs) (1.89–8.81%), which confirmed the applicability of the sensor in practical applications. This study provides a new analytical method for measuring the level of GPC3 in the early diagnosis of HCC. Full article