There is an increasing interest in Electrochemical Impedance Spectroscopy (EIS) as the transducing method for label-free biosensors. This technique allows the direct detection of the affinity complex formation through the variation of the capacitance or that of the charge transfer resistance at the biofunctionalized electrode/biological liquid interface. This chapter will present the physical principles of EIS, the different designs of the micro/nanoelectrodes, the new ways for the elaboration of the recognition biofilm, the performances of detection of the different biotargets, and the perspectives for the in situ detection.