A novel electrochemical immunosensor for testosterone based on direct attachment of antitestosterone on hybrid gold nanoparticles/carbon nanotubes-Teflon composite electrodes is reported. This material combined the ability of gold nanoparticles for biomolecules immobilization retaining their biological activity with synergic electrocatalytic effect demonstrated by coupling gold nanoparticles and carbon nanotubes. Competitive assay between testosterone and HRP- testosterone for binding sites of antibodies was employed. Amperometry at −0.05 V vs. Ag/AgCl was used to monitor affinity reaction upon addition of H2O2 with catechol as redox mediator. Calibration plot was linear between 0.1 and 10 ng/mL testosterone. LOD value was 85±6 pg/mL. Good results were obtained in the application to spiked human serum.