The yeast Saccharomyces cerevisiae (S. cerevisiae) is considered an ideal eukaryotic model and has long been recognized for its pivotal role in numerous industrial production processes. Depending on the cell cycle phases, microenvironment, and species, S. cerevisiae varies in shape and has different sizes of each shape such as singlets, doublets, and clusters. Obtaining high-purity populations of uniformly shaped S. cerevisiae cells is crucial in fundamental biological research and industrial operations. In this study, we propose an acoustofluidic method for separating S. cerevisiae cells based on their size using surface acoustic wave (SAW)-induced acoustic radiation force (ARF). The SAW-induced ARF increased with cell diameter, which enabled a successful size-based separation of S. cerevisiae cells using an acoustofluidics device. We anticipate that the proposed acoustofluidics approach for yeast cell separation will provide new opportunities in industrial applications.