Obtaining a purified male sperm specimen from the original forensic sample mixtures is a critical procedure in identifying the criminal suspect in the forensic analysis of sexual assault crimes. Differential extraction (DE) has been used as the gold standard for extracting DNA preferentially from sexual assault samples, however it is laborious, time-consuming and inefficient. In this report a continuous flow two-step acoustic cell separation scheme is proposed for the removal of cell debris and cell-free genomic contaminants, followed by separation of sperm cells from epithelial cells. Based on the above method, a portable forensic sperm cell purification instrument has been constructed for on-site pretreatment of forensic samples in sexual assault crimes, where removal of contaminants and isolation of sperm cells could be automatically performed without the use of cell lysis buffer.