Polymer-based substrates with patterned microstructure on the surfaces, e.g., cell culturing scaffolds, have been utilized in biomedical applications. This paper develops a novel method to fabricate the localized microstructure on the polymer-based substrate with the assistance of standing surface acoustic wave (SAW) and user-defined acoustic waveguides. The specific designed acoustic waveguides can localize the standing acoustic waves and transmit to the liquid film and excite patterned microstructures on the surface, then using ultraviolet (UV) to solidify the substrate with patterned microstructures. The structural design and fabrication of the SAW device and three different shaped acoustic waveguides are presented. Then, experimental setup and procedures to verify the polymer-substrate with localized microstructures fabrication are performed. By using the different shape of the acoustic waveguides, several types of patterned microstructures with different morphologies are successfully fabricated. Results demonstrated that the proposed fabrication method is an effective way to fabricate polymer-based substrate with localized patterned microstructures, which may have potential in the research on tissue engineering, cell-cell interaction, and other biomedical applications.