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Spatial distribution of biological cells plays a key role in tissue engineering for reconstituting the cellular microenvironment, and recently, acoustofluidics are explored as a viable tool for controlling structures in tissue fabrication because of its good biocompatibility, low-power consumption, automation capability, nature of non-invasive, and non-contact. Herein, a reusable acoustofluidic device is developed using surface acoustic waves for manipulating microparticles/cells to form a 3D matrix pattern inside a scaffold-based hydrogel contained in a millimetric chamber. The 3D patterned and polymerized hydrogel construct can be easily and safely removed from the chamber using a proposed lifting technique, which prevent any physical damages or contaminations and promote the reusability of the chamber. The generated 3D patterns of microparticles and cells are numerically studied using a finite-element method, which is well validated by the experimental results. The proposed acoustofluidic device is a useful tool for in vitro engineering 3D scaffold-based artificial tissues for drug and toxicity testing and building organs-on-chip applications. Ministry of Education (MOE) Nanyang Technological University The authors gratefully acknowledge the support of 1) Nanyang Technological University and the Ministry of Education of Singapore through a Ph.D. Scholarship and AcRF Tier 1 research grant (RG 96/18); 2) the UK Engineering and Physical Sciences Research Council (EPSRC) grants (EP/P018998/1); and 3) Special Interesting Group of Acoustofluidics funded by UK Fluids Network (EP/N032861/1).