The ultrasonic actuator can be used in medical applications because it is label-free, biocompatible, and has a demonstrated history of safe operation. Therefore, there is an increasing interest in using an ultrasonic actuator in the non-contact manipulation of micromachines in various materials and sizes for therapeutic applications. This research aims to design, fabricate, and characterize a single-sided transducer array with 56 channels operating at 500 kHz, which provide benefits in the penetration of tissue. The fabricated transducer is calibrated using a phase reference calibration method to reduce position misalignment and phase discrepancies caused by acoustic interaction. The acoustic fields generated by the transducer array are measured in a 300 mm × 300 mm × 300 mm container filled with de-ionized water. A hydrophone is used to measure the far field in each transducer array element, and the 3D holographic pattern is analyzed based on the scanned acoustic pressure fields. Next, the phase reference calibration is applied to each transducer in the ultrasonic actuator. As a result, the homogeneity of the acoustic pressure fields surrounding the foci area is improved, and the maximum pressure is also increased in the twin trap. Finally, we demonstrate the capability to trap and manipulate micromachines with acoustic power by generating a twin trap using both optical camera and ultrasound imaging systems in a water medium. This work not only provides a comprehensive study on acoustic actuators but also inspires the next generation to use acoustics in medical applications.