Purpose: Particle separation in solution is one of important process in a unit operation as well as in an extract preparation for biosensors. Contrary to centrifuge-type of mesh-type filter, using an ultrasonic standing wave make the filtering process continuous and free from maintenance. It is needed to investigate the characteristics of particle movement in the ultrasonic standing wave field. Methods: Through the computer simulation the effects of major design and driving parameters on the alignment characteristics of particles were investigated, and a cylindrical chamber with up-stream flow type was devised using two circular-shape PZTs on both sides of the chamber, one for transmitting ultrasonic wave and the other for just reflecting it. Then, the system performance was experimentally investigated as well. Results: The speed of a particle to reach pressure-node plane increased as the acoustic pressure and size of particle increased. The maximum allowable up-stream flow rate could be calculated as well. As expected, exact numbers of pressure-node planes were well formed at specific locations according to the wavelength of ultrasonic wave. As the driving frequency of PZT got close to its resonance frequency, the bands of particles were observed clearer, which meant the particles were trapped into narrower space. Higher excitation voltages to the PZT produced a greater acoustic force with which to trap particles in the pressure-node planes, so that the particles gathered could move upwards without disturbing their alignments even at a higher inlet flow rate. Conclusions: This research showed the feasibility of particle separation in solution in the continuous way by an ultrasonic standing wave. Further study is needed to develop a device to collect or harvest those separated particles.