Polymer brushes are frequently used as surface-tethered antifouling layers in biosensors to improve sensor surface-analyte recognition in the presence of abundant non-target molecules in complex biological samples by suppressing nonspecific interactions. However, because brushes are complex systems highly responsive to changes in their surrounding environment, studying their properties remains a challenge. Electrochemical impedance spectroscopy (EIS) is an emerging method in this context. In this mini review, we aim to elucidate the potential of EIS for investigating the physicochemical properties and structural aspects of polymer brushes. The application of EIS in brush-based biosensors is also discussed. Most common principles employed in these biosensors are presented, as well as interpretation of EIS data obtained in such setups. Overall, we demonstrate that the EIS-polymer brush pairing has a considerable potential for providing new insights into brush functionalities and designing highly sensitive and specific biosensors.