To obtain individual Y-short tandem repeat (STR) profiles in a multi-suspect sexual assault case. Methods. We used laser cut microdissection to capture the single sperm cell in the multi-contributor semen sample, combined with the low volume polymerase chain reaction (LV-PCR) method to genotype the single sperm cell profiles using the Yfiler(®) kit. Consensus DNA profiles were generated from 5 replicate experiments. Results. Ninety-four parallel LV-PCRs were performed and 41 reactions (44%) produced Y-STR profiles with more than nine loci. Three individual Y-STR profiles were successfully obtained. Conclusion. The three Y haplotype units matched three known perpetrators' genotypes. Our results showed that single sperm cells Y-STR analysis was a powerful method for analyzing multi-donor semen mixture sample.