<p>Horizontal focusing, which precisely aligns cells within the focal plane of microscopic imaging, is essential for ensuring the analytical accuracy and reliability of imaging microflow cytometry. Hydrodynamic focusing, dependent on sheath flows, is constrained by parabolic velocity profiles that induce uneven shear forces, limiting lateral alignment precision. Active methods, such as acoustic focusing, enhance particle positioning but often face challenges in complexity and scalability. In contrast, viscoelastic focusing provides a simple and scalable alternative by leveraging the viscoelastic properties of non-Newtonian fluids. To optimize its application in microflow cytometry, we systematically investigate horizontal focusing performance depending on the effects of polymer molecular weight, concentration, and flow rate. Our findings reveal that stable single-plane focusing requires a fine balance between elastic and inertial forces, as higher molecular weight polymers generate stronger viscoelastic forces but may induce multiple equilibrium positions due to significant shear-thinning effects, disrupting single-plane alignment. These results highlight the importance of fine-tuning polymer properties and flow conditions to achieve precise and reliable horizontal focusing for microflow cytometry applications.</p>