Microparticle separation has demonstrated significant potential for biological, chemical, and medical applications. We introduce a surface acoustic wave (SAW)-based microfluidic device for separation of elastic and rigid microspheres based on their property and size. By tuning the SAWs to match the resonant frequencies of certain microspheres, those particles could be selectively separated from the other microspheres. When microspheres are exposed to an acoustic field, they experience the SAW-induced acoustic radiation force (ARF), whose magnitude is dependent on the microparticle size and properties. We modeled the SAW-induced ARF based on elastic sphere theory and conducted a series of experiments to separate elastic and rigid microspheres. We further utilized the acoustofluidic method for the separation of Thalassiosira Eccentrica microalgae based on the differences in their sizes with purity exceeding 90%. We anticipate that our technique will open up new possibilities for sample preparation, detection, and diagnosis in various emerging biological and medical analyses.