A novel and sensitive label-free electrochemical immunosensor was developed for enzyme-free determination of biomarker tumor necrosis factor a antigen (TNF-alpha) based on entrapping of a TNF-alpha antibody onto nanocomposite containing fullerene-functionalized multiwalled carbon nanotubes and ionic liquid (1-butyl-3-methylimidazolium bis (trifluoromethyl sulfonyl)imide) (C-60-CNTs-IL). C-60-CNTs-IL was used as a desirable sensor platform and excellent electrocatalyst for oxidation of catechol as a probe in an immunosensor. Determination of TNF-alpha was based on its obstruction of the electrocatalytic oxidation of catechol by C-60-CNTs-IL after binding to the surface of an electrode through interaction with the anti-TNF-alpha. The protein layers on the modified electrode generated a barrier for electrontransfer; we demonstrated the effect of immobilized protein layers and modification of the electrode on the kinetic parameters in every step of immunosensor construction. Various kinetic parameters such as electrode surface area (A), diffusion coefficient (D) and electron-transfer rate constant (k(s)) were also studied and calculated in the aqueous solution. Under the optimal conditions, the results demonstrated that this electrochemical immunosensor had a dynamic range of between 5.0 pg/mL to 75 pg/mL with a low detection limit of 2.0 pg/mL for TNF-alpha. The fabricated immunosensor was successfully applied to the determination of TNF-alpha in serum samples. The obtained results showed the change of kinetic parameters due to the effect of each step of immunosensor fabrication and the fabricated immunosensor demonstrates the promising applications in protein diagnostics and bioassay. (C) 2015 Elsevier B.V. All rights reserved.