In the pursuit of refining the fabrication of three-dimensional (3D) microelectrode arrays (MEAs), this study investigates the application of ultrasonic vibrations in template-assisted electrodeposition. This was driven by the need to overcome limitations in the deposition rate and the height uniformity of microstructures developed using conventional electrodeposition methods, particularly in the field of in vitro electrophysiological investigations. This study employs a template-assisted electrodeposition approach coupled with ultrasonic vibrations to enhance the deposition process. The method involves utilizing a polymeric hard mask to define the shape of electrodeposited microstructures (i.e., micro-pillars). The results show that the integration of ultrasonic vibrations significantly increases the deposition rate by up to 5 times and substantially improves the uniformity in 3D MEAs. The key conclusion drawn is that ultrasonic-enhanced template-assisted electrodeposition emerges as a powerful technique and enables the development of 3D MEAs at a higher rate and with a superior uniformity. This advancement holds promising implications for the precision of selective electrodeposition applications and signifies a significant stride in developing micro- and nanofabrication methodologies for biomedical applications.