Two excitation methods based on 2D normal mode were proposed using two piezoelectric transducers (PZTs) with opposite phase and in-phase to excite the two dimensional normal modes of (1, 1) and (2, 1), respectively. The theoretical models of the excitation modes were deduced by the wave equation. An ultrasonic separator was built and the acoustic simulation of the ultrasonic excitation modes was modeled in Ansys software. The simulation results of the sound pressure distribution in the flow channel show that (1, 1) and (2, 1) are successfully excited. The micro-separator with 8 mm width and 0.2 mm high flow channel was fabricated on silicon on insulator (SOI) by micro-processing technology to form a perfect reflection layer of the separator. An experimental platform was established and the results show that both excitation methods can achieve the separation of suspended particles with high-through of 100 μL/min. In the experiment of (1, 1) excitation method, it can be seen that most of the particles converge in the center of the separation cavity into a large bunch, and in the experiment of (2, 1) excitation method, the particles mainly converge on both sides of the separation chamber. Both methods can successfully separate suspended particles out of the fluid.