Obtaining a single male DNA profle from aged mixed stains composed of sperm and vaginal epithelial cells, which are common challenging forensic biological materials, is difficult due to severe cell degradation and trace sperm content. This paper presents the testing process and successful experience of a sexual assault case, providing a promising solution for handling aged mixed stains with limited sperm content in practical scenarios. The sample was a vaginal swab from the victim, which yielded an unidentifed single male DNA profle by DNA analysis in 2004. In the identification process in 2023, the PSA test on the sample was confrmed to be positive. Only the supernatant part yielded a single female DNA profle and an incomplete Y-STR profle using the conventional differential lysis method, and no DNA was detected in the precipitate part. In contrast, by utilizing commercial immunomagnetic bead kits and optimized experimental procedures, sperm cells were successfully isolated from the seminal and vaginal mixed-stain samples under enhanced incubation conditions, resulting in the acquisition of a single male DNA profle. At the end of this paper, the application value and precautions of the method for sperm cell separation based on immunomagnetic beads in the analysis of mixed stains are discussed.