A bubble-based acoustofluidic filtration (BAF) microfluidic device, which employs cross-flow filtration (CFF) and acoustic streaming, separates cells with high efficiency for forensic analysis. Forensic samples are typically complex and contain a substantial number of squamous epithelial cells from the female vagina, which tend to have fouling problems during filtration due to their morphological and cell adhesion differences. To overcome this issue, the BAF device utilizes bubble oscillation by bulk acoustic wave (BAW) to generate acoustic streaming, which offers additional hydrodynamic forces for side flushing cleaning and achieves effective removal within a mere 0.5 seconds. Our device is tested with imbalanced cell mixtures of sperm and epithelial cells with large disparity ratios. By concurrently employing CFF and acoustic streaming, the samples with our sperm-enrichment can achieve 91.72-97.78% for the recovery rate and 74.58-89.26% for the purity in the sperm enrichment. They are further subjected to short tandem repeat (STR) profiling, enabling the identification of perpetrators. Notably, even samples with minimal sperm cells demonstrated a significant increase in the male donor DNA ratio, while the peak heights of female alleles became virtually undetectable. The exceptional cell separation capability demonstrated by our BAF device highlights its potential applications in forensic sciences and other areas of cell biology.