Two-dimensional concentric asymmetric microelectrodes play a crucial role in developing sensitive and specific biological assays using fluid micromixing generated by alternating current electrohydrodynamics (ac-EHD). This paper reports the design, simulation, fabrication, and characterization of fluid motion generated by 3D concentric microelectrodes for the first time. Electric field simulations are used to compare electric field distribution at the electrodes and to analyze its effects on microfluidic micromixing in 2D and 3D electrodes. Three-dimensional devices show higher electric field peak values, resulting in better fluid micromixing than 2D devices. As a proof of concept, we design a simple biological assay comprising specific attachment of streptavidin beads onto the biotin-modified electrodes (2D and 3D), which shows ~40% higher efficiency of capturing specific beads in the case of 3D ac-EHD device compared to the 2D device. Our results show a significant contribution toward developing 3D ac-EHD devices that can be used to create more efficient biological assays in the future.