Acoustic force patterning is an emerging technology that provides a platform to control the spatial location of cells in a rapid, accurate, yet contactless manner. However, very few studies have been reported on the usage of acoustic force patterning for the rapid arrangement of biological objects, such as cells, in a three-dimensional (3D) environment. In this study, we report on a bio-acoustic force patterning technique, which uses surface acoustic waves (SAWs) for the rapid arrangement of cells within an extracellular matrix (ECM)-based hydrogel such as gelatin methacryloyl (GelMA). A proof-of-principle was achieved through both simulations and experiments based on the in-house fabricated piezoelectric SAW transducers, which enabled us to explore the effects of various parameters on the performance of the built construct. The SAWs were applied in a fashion that generated standing SAWs (SSAWs) on the substrate, the energy of which subsequently was transferred into the gel, creating a rapid, yet contactless alignment of the cells (< 10 seconds, based on the experimental conditions). Following UV radiation induced photo-crosslinking of the cell encapsulated GelMA pre-polymer solution, the patterned cardiac cells readily spread after alignment in the GelMA hydrogel and demonstrated beating activity in 5-7 days. The described acoustic force assembly method can be utilized not only to control the spatial distribution of the cells inside a 3D construct, but can also preserve the viability and functionality of the patterned cells (e.g. beating rates of cardiac cells). This platform can be potentially employed in a diverse range of applications, whether it is for tissue engineering, in vitro cell studies, or creating 3D biomimetic tissue structures.