Recently, the technology integrating acoustic with microfluidics, also called acoustofluidics, has drawn significant attention based on its unique advantages through the combinations of solids and fluids, and the interplay of mechanics and electronics. Despite its promise to address many challenges in biomedical research, it's still in its infancy. Much work needs to be done to fully explore the potential of acoustofluidics as an everyday tool for real-world applications. To achieve this goal, many challenging problems related to physics, engineering, and biomedical applications of acoustofluidics need to be solved. In this dissertation, I systematically investigated the pivotal components in SAW-based acoustofluidics and developed novel acoustofluidic devices with innovative configurations. The main works involved in this dissertation are as follows:First, we explore a fully integrated chip-scale artificial graphene-like piezo-optomechanical structure and investigate the characteristics of surface phonon transport in it. We exploit the surface phonon polariton (SPhP) coupling between electromagnetic (EM) waves and atomic-level vibrations in the piezo-optomechanical metamaterial to create a SAW bandgap. We monolithically integrate the metamaterial with a lithium niobate substrate through a non-destructive and ad-hoc electric field poling technique. By merging the metamaterial with an acoustofluidic system, we present the first experimental demonstration of both phase and amplitude coherent scattering of SAW by monitoring the response of micrometer-sized non-organic particles to the resulting SAW field established in the microfluidic channel. Then, we present an integrated phononic crystal acoustofluidic device (IPAD) that incorporates phononic crystal into a SAW-based acoustofluidic system to achieve a multi-functional acoustofluidic platform. To perform band structure tuning, we systematically investigate the band gap properties of 2D SPhP phononic crystals and analyzed the band gap variation with different filling ratios. With the assistance of the frequency domain engineering technique, we integrate multiple SAW schemes to perform multi-operations by merging standing SAW-induced particle alignment and traveling SAW-induced off-center particle translocation. We experimentally demonstrate the ability of the IPAD platform to perform particle separation of different sizes with a better separation efficiency and higher throughput than one single SAW scheme without compromising the device’s compactness. In the third part (Chapter 4), we develop a Two-Stage Acoustic Focusing Compressibility Cytometry (AF-CC) technique that merges standing surface acoustic wave (SSAW)-based cell focusing and SSAW-based off-center cell translocation within a single microfluidic channel in a cascaded manner. By three-dimensional (3D) accurately tight-focusing cells into a single line, we can achieve real-time quantification of whole-cell compressibility by monitoring the trajectories of suspended cells. A one-to-one mapping of cell compressibility to the cell transit path length over its trajectory is established, allowing us to realize in-situ and high-throughput (~ 10 cells/second) quantitative analysis of single-cell. We demonstrate that AF-CC technique is sensitive enough to precisely distinguish the compressibility of three distinct murine hematopoietic cell types and their inhibitor-treated counterparts with cytoskeleton-perturbing molecules. In the final part of the thesis (Chapter 5), we present a label-, contact-, flow perturbation-free acoustic drifting effect (ADE) technology significantly boosts the advective transport of suspended colloids and biological cells across the channel in a remarkably short distance. Our ADE platform uses a simple and fabrication-friendly configuration by integrating a straight microfluidic channel with a pair of metallic interdigitated transducers (IDTs) lithographically deposited on a LiNbO3 substrate. By exploiting the well-engineered acoustic field, this ADE platform allows us to selectively separate target particles/cells and achieve immunoaffinity-based isolation. Our platform is proven to efficiently translate the target particles to the functionalized bottom of the microfluidic channel by the exerted downward acoustic radiation force, where the immobilized capture agents can bind with the target particles. Finally, We also demonstrate the selective capture of rare cells (T lymphocyte cells) with a 90% separation efficiency at a 1 mL/hr flow rate, comparable to current rare cell isolation technologies.