To address the challenge of focusing small-diameter microorganisms, such as bacteria and yeast, in flow cytometry, we conducted a study on microfluidic chips for microorganism focusing. A novel square channel was developed based on traditional straight-channel designs. A finite element model of the square-channel microfluidic chip was established to enable the optimization of structural parameters, including the channel width and length, through finite element simulations of fluid flow and particle trajectories. The simulation showed that when the channel width was 20 μm, and the unit length was 80 μm, particles were focused within 63% of the channel. A silicon-based substrate was used to fabricate the square-channel microfluidic chip, and a prototype flow cytometer was constructed to test it. The experimental results indicate that with a total channel length of approximately 2.16 cm, the actual focusing range for yeast cells reached 70%, thus satisfying the design requirements for yeast focusing.