Frequency-dependent electrochemical impedance spectroscopy has been used to characterize the changes in electrical response that accompany specific binding of a protein to its substrate, using the biotin-avidin system as a model. Our results show that avidin, at concentrations in the nanomolar range, can be detected electrically in a completely label-free manner under conditions of zero average current flow and without the use of any auxiliary redox agents. Impedance measurements performed on biotin-modified surfaces of gold, glassy carbon, and silicon were obtained over a wide frequency range, from 5 mHz to 1 MHz. On each biotin-modified surface, binding of avidin is most easily detected at low frequencies, <1 Hz. Electrical circuit modeling of the interface was used to relate the frequency-dependent electrical response to the physical structure of the interface before and after avidin binding. Electrical measurements were correlated with measurements of protein binding using fluorescently labeled avidin.