Acoustofluidics, an interdisciplinary nexus of microfluidics and acoustics, is propelling the critical functionalities of manipulation, separation, and mixing within microscale environments. This integration leverages the accuracy of microfluidics with the manipulation capabilities of acoustics, thereby enhancing the vital sample processing steps and satisfying inquiries in experiments. To fulfill the requisites of practical application in clinical and research arenas, the evolution of acoustofluidics concentrates on accomplishing finer particle separation, instantaneous manipulation, and augmented integration capacity. Acoustofluidics has evolved into a sophisticated and versatile instrument for handling specimens and reactants, prompting a trend towards devices characterized by stable performance at elevated frequencies, programmable control, and seamless integration with auxiliary microfluidic systems. In this review, we present the latest advancements in the development of sophisticated acoustofluidic systems that enhance efficiency and enable precise modulation of performance across spatial and temporal scales, thereby extending their functionality and suitability for practical applications.