In this work, we demonstrate a sheathless acoustic fluorescence activated cell sorting (aFACS) system by combining elasto-inertial cell focusing and highly focused traveling surface acoustic wave (FTSAW) to sort cells with high recovery rate, purity, and cell viability. The microfluidic sorting device utilizes elasto-inertial particle focusing to align cells in a single file for improving sorting accuracy and efficiency without sample dilution. Our sorting device can effectively focus 1 μm particles which represents the general minimum size for a majority of cell sorting applications. Upon the fluorescence interrogation at the single cell level, individual cells are deflected to the target outlet by a ∼50 μm wide highly focused acoustic field. We have applied our aFACS to sort three different cell lines (i.e., MCF-7, MDA-231, and human-induced pluripotent stem-cell-derived cardiomyocytes; hiPSC-CMs) at ∼kHz with a sorting purity and recovery rate both of about 90%. A further comparison demonstrates that the cell viability drops by 35-45% using a commercial FACS machine, while the cell viability only drops by 3-4% using our aFACS system. The developed aFACS system provides a benchtop solution for rapid, highly accurate single cell level sorting with high cell viability, in particular for sensitive cell types.