This work demonstrates a standing wave focusing method to complete twodimension and sheathfree focusing of particles for flow cytometer. Cells or beads in a volume of less than 82 μL are forced to cross the detection area one by one under a flow rate higher than 0.5 mL/min, which can improve the analysis accuracy and realize undiluted sample recovery. The principle of standing wave formation, acoustic resistance matching and the particle force equilibrium are discussed, and the motion trajectory of particle under different parameters is simulated. Referring to this model, an acoustic focusing experimental platform driving in a frequency of 1.462 MHz is built to verify the feasibility and performance by polystyrene beads in diameters of 10 and 20 μm. The results showed that beads in random distribution can be focused into a plane, and more tightly focusing can be achieved at low flow rate and high driving voltage. It is proved that the bigger beads are focused more easily than the smaller ones. All the experimental results coincides well with the simulation.