Objectives To establish a sensitive and special method for the detection of Ureaplasma urealyticum(Uu) using PCR microplate hybridization (PCR MPH). Methods A primer of ureasea gene was labeled by biotin. The amplification product was captured on streptavidin coated microplates, then products were quantified by hybridization with a digoxigenin labeled internal oligonucleotide probe. After revelation with an anti digoxigenin alkaline phosphatase coupled antibody(anti DIG AP), the amount was determined by optical reading. At the same time, PCR MPH was compared with Bio Merieux Mycosplasma IST. Results A method of PCR MPH for detecting Uu DNA was established. The morbidity among three groups for detecting 158 clinical samples was analysed. 65 were detected by PCR MPH and 56 by culture.Conclusion The results showed that this assay is rapid, sensitive, specific, and accurate, and is of value in clinical therapy.