An ultrasensitive electrochemical immmunosensor was demonstrated to be capable of detecting the hormone cortisol down to concentrations as low as 16 pg mL-1. In addition, the immunosensor displayed a sensitivity of 1.6 μA pg-1 mL-1 and a linear range up to ∼2500 pg mL-1 of cortisol. This immunosensor was constructed based on a Au nanoparticledimethyl 3,3′-dithiobispropionimidate·2HCl (DTBP)-Protein G scaffold-modified Au electrode. In this work, the Au nanoparticles were used to increase the electrochemically active surface area by 28% (with a standard deviation of 3%) to enhance the quantity of the Protein G scaffold on the electrode. Thiolation of Protein G by DTBP aided in avoiding the confirmation change of Protein G, while this Protein G-DTBP component offered an orientation-controlled immobilisation of the capture antibody on the Au electrode. In this immunosensor, a monoclonal anti-cortisol capture antibody was optimally aligned by the scaffold before a competitive immunoassay between sample cortisol and a horseradish peroxidase-labelled cortisol conjugate was conducted. For quantitative analysis, square wave voltammetry was used to monitor the reduction current of benzoquinone produced from a horseradish peroxidase catalysed reaction. The improved analytical performance of our immunosensor was attributed to the synergetic effect of Au nanoparticles and the Protein G-DTBP scaffold.