Polymer-based substrate with region-selective microstructures are crucial for many biomedical applications. Here, we explored a novel method based on standing surface acoustic waves (SSAWs) for the fabrication of localized polymer-based microstructures via a predefined waveguide. When the SSAWs are excited, the generated acoustic pressure field can be controlled in a predetermined region of the fluid surface through controlling the size and shape of the waveguide geometry. On the basis of the capillary wave motion, the generated acoustic pressure field can excite microwavy patterned structures on the surface. Then with use of ultraviolet (UV) solidification, the polymer-based substrates with region-selective patterned microstructures can be successfully fabricated. Both finite element modeling and experimental studies demonstrated that the polymer substrate with different region-selective microstructures can be achieved by selecting the pairs of interdigital transducers (IDTs) and shapes of the predefined waveguides. The results showed that the proposed method is effective for fabricating polymer-based substrate with region-selective microstructures and may have potential in cell-laden chips for tissue engineering, cell-cell interactions, and other biomedical applications.