Advances in nanomaterials, combined with electrochemical impedance spectroscopy (EIS), have allowed electrochemical biosensors to have high sensitivity while remaining label-free, enabling the potential for portable diagnosis at the point-of-care. We report porous, 3D vertically aligned carbon nanotube (VACNT) electrodes with underlying chromium electrical leads for impedance-based biosensing. The electrodes are characterized by electrode height (5, 25, and 80 μm), gap width (15 and 25 μm), and geometry (interdigitated and serpentine) using scanning electron microscopy, cyclic voltammetry, and electrochemical impedance spectroscopy (EIS). The protein streptavidin is functionalized onto VACNT electrodes for detection of biotin, as confirmed by fluorescence microscopy. EIS is used to measure the change in impedance across electrodes for different biotin concentrations. The impedance data shows two distinct semi-circular regions which are modeled by an equivalent electrical circuit. VACNT electrode height, gap width, and geometrical pattern each have an impact on sensor sensitivity, with tall, closely-spaced VACNT interdigitated electrodes (IDEs) having the highest sensitivity. With an electroactive surface area that is 15x the 2D geometric area, 80 μm tall VACNT IDEs with a gap width of 15 μm are 4.3x more sensitive than short (5 μm) IDEs and 1.6x more sensitive than serpentine electrodes (SEs). The biosensors obtain a limit of detection of 1 ng/mL biotin with two linear sensing regions (0.001 – 1 μg/mL and 1 – 100 μg/mL). Although this biosensing platform is shown with streptavidin and biotin, it could be extended to other proteins, antibodies, viruses, and bacteria.