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Ultrasound is gaining attention as an alternative tool to regulate chemical processes due to its advantages such as high cost-effectiveness, rapid response, and contact-free operation. Previous studies have demonstrated that acoustic bubble cavitation can generate energy to synthesize functional materials. In this study, we introduce a method to control the spatial distribution of physical and chemical properties of hydrogels by using an ultrasound-mediated particle manipulation technique. We developed a surface acoustic wave device that can localize micro-hydrogel particles, which are formed during gelation, in a hydrogel solution. The hydrogel fabricated with the application of surface acoustic waves exhibited gradients in mechanical, mass transport, and structural properties. We demonstrated that the gel having the property gradients could be utilized as a cell-culture substrate dictating cellular shapes, which is beneficial for interfacial tissue engineering. The acoustic method and fabricated hydrogels with property gradients can be applied to design flexible polymeric materials for soft robotics, biomedical sensors, or bioelectronics applications.