Highly sensitive and selective biosensors for accurate determination of specific protein biomarkers at low levels in serum are a prerequisite for the present healthcare systems. Therefore, here we developed a label-free impedimetric method for tumor necrosis factor-alpha (TNF-α) detection using reduced graphene oxide (RGO) with gold nanoparticles (AuNP) on an indium tin oxide (ITO) microdisk electrodes. The detection mechanism relies on resistance change occurs due to [Fe(CN)6]3−/4− redox probe movement towards the conductive channels of the AuNP-RGO films gated by the recognition of the target biomarker by its anti-TNF-α antibody. The conductivity of the AuNP-RGO structures enhanced by 10-fold in comparison with bare electrode chips. The observed resistance changes at 2 Hz (ΔR at 2Hz) enabled the quantification of various concentrations of TNF-α in human serum (CHS-TNF-α). The antibody-adsorbed electrode showed a good increment of resistance change (ΔR) with additions of antigen concentration. The sensor possesses a linear range of 1–1000 pg/ml had a detection limit of 0.67 pg/ml (38.51 fM) and 0.78 pg/ml (44.83 fM) in PBS and human serum, respectively.