Blastocystis hominis is a common enteric parasite of worldwide distribution. Its pathogenetic potential has not yet been established, although numerous case reports suggest that B. hominis may cause the development of various gastrointestinal symptoms and disorders. The detection of the parasite in stool specimens is conventionally done by microscopy of direct smears, fecal concentrates, or permanently stained smears; however, morphology-based diagnosis is problematic. The aim of this study was to develop and evaluate a polymerase chain reaction (PCR) technique for the direct detection of B. hominis in human stool samples. Primers were based on small subunit ribosomal DNA and able to detect ≥32 parasites/200 mg stool artificially spiked with cultured B. hominis. In the evaluation of 43 clinical specimens, the PCR was tested against the formol ethyl acetate concentration technique (FECT) and a culture technique, proving 100% test specificity and a significantly higher sensitivity than the FECT. The PCR method is recommended for screening clinical specimens for B. hominis infection and for use in prevalence studies.