Avidin-biotin has been controllable immobilized on the surface of gold electrodes using mercaptopropionic acid as self-assembled monolayer. Electrochemical impedance spectroscopy (EIS) was employed to investigate the changes that appear at the electrode surface in the presence of a redox mediator, K3[Fe(CN)6]. An electrical model more complex than that in other studies was used to interpret the EIS measurements (Randles circuit). This model is very useful because it takes into consideration both the transfer of electrons at the electrode/electrolyte interface and the diffusion of redox species through the double layer. The model allowed us to determine some important parameters like solution resistance Rsol, charge-transfer resistance Rct, double-layer capacitance Cdl, Warburg resistance RW, and the diffusion time constant τ. The EIS results proved that immobilization of avidin-biotin increased the charge-transfer resistance Rct, due to the insulating character of these molecules.