Picomolar electrochemical detection of human immunodeficiency virus type-1 protease (HIV-1 PR) using ferrocene (Fc)-pepstatin-modified surfaces has been presented. Gold electrode surface was modified with gold nanoparticles (AuNP) or thiolated single walled carbon nanotubes/gold nanoparticles (SWCNT/AuNP). Thiol-terminated Fc-pepstatin was then self-assembled on such surfaces as confirmed by Raman spectroscopy and scanning electron microscope. The interaction between the Fc-pepstatin-modified substrates and HIV-1 PR was studied by cyclic voltammetry and electrochemical impedance spectroscopy. Both electrode materials showed enhanced electrochemical responses to increasing concentrations of HIV-1 PR with shifting to higher potentials as well as decrease in the overall signal intensity. However, the sensing electrode modified with thiolated SWCNTs/AuNPs showed remarkable detection sensitivity with an estimated detection limit of 0.8 pM.