A practical and novel enzyme-free sandwich-type electrochemical aptasensor for sensitive detection of protein biomarker tumor necrosis factor-alpha (TNF-alpha) was designed by using bimetallic Ag@Pt core-shell nanoparticles functionalized graphene nanosheets (Ag@Pt-GRs) as novel desirable labels for signal amplification and Au nanoparticles functionalized graphene/chitosan (Au-GRs/CS) nanocomposite as sensing platform. The results showed that Ag@Pt-GRs nanocomposite exhibited attractive electrocatalytic activity without any enzyme toward the oxidation of catechol as analytical signal and also yielded large surface area, which improve the amount of immobilized aptamer and leading to the formation of Ag@Pt-GRs/apt bioconjugate. The aptasensor was constructed by modifying screen-printed electrodes with Au-GRs/CS nanocomposite which could provide a large electroconductive surface area to immobilize a large amount of aptamers, resulting in significant improvement in the sensitivity of aptasensor. The proposed electrochemical aptasensor showed a dynamic range from 5.0 pg/mL to 70 pg/mL with a low detection limit of 1.64 pg/mL for TNF-alpha. The analytical usefulness of the aptasensor was finally demonstrated analyzing serum samples. The main advantages of this aptasensor are the simple fabrication method, high sensitivity, specificity, good reproducibility and stability as well as acceptable accuracy for TNF-alpha detection in human serum, which might have broad applications in protein detection and disease diagnosis. (C) 2016 The Electrochemical Society. All rights reserved.