Microfluidic device with patted microstructures on the substrate surface was used to regulate cell adhesion, morphology, and functions in tissue engineering. We developed a microfluidic device which employing microscale patted microstructures to achieve enhanced cell adhesion and migration. Biocompatible hydrogel substrates with micro-wavy and lattice-patted microstructures were fabricated using standing surface acoustic waves and ultraviolet solidification. After seeding the L929 mouse fibroblast cells onto the patted substrate of the microfluidic device, we determined that the viability and proliferation rate of cell migration can be greatly enhanced. Furthermore, L929 cells showed two types of gathering modes after 48 h of culturing. Cell growth was guided by the patted substrate used in the microfluidic device and showed differences in the location distribution. Therefore, the developed microfluidic device with patted microstructures can extend the application of in vitro cell culturing for future drug development and disease diagnosis.