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3D self-assembly of biological tissues is essential for organ development, tissue engineering, transplantation, and organ regeneration. Although multicellular spheroids have been broadly used as 3D tissue models, it is challenging to precisely manipulate their interaction, limiting their potential in understanding and modeling complex tissue self-assembly. Here, we present a novel acoustofluidic device for the controllable fusion of multicellular spheroids. By introducing standing sound waves into a microfabricated chamber, our acoustofluidic device could achieve the trapping and assembling of multicellular spheroids in a highly biocompatible, label-free, and contact-free manner. After incorporating with a time-lapse imaging and in situ culture approach, our acoustofluidic system could manipulate and track the dynamic interaction of the multicellular spheroids. Moreover, the interaction dynamics of homotypic and heterotypic spheroid pairs were quantitatively characterized, and the adhesion molecules of paired spheroids were analyzed. Together, we developed a unique engineering tool, which is contactless, versatile, and biocompatible, all of which make it useful for 3D biological self-assembly and translational applications in regenerative medicine. [ABSTRACT FROM AUTHOR]