Abstract: A double polymerase chain reaction (PCR) was developed for simultaneous detection and germ group classification of bacteria and yeasts of bovine mastitis. We developed a new method that extracts both bacteria and yeast DNA from milk using β-mercaptoethanol, which can decompose cell walls. At the same process we used lysozyme and snailase, which can assimilate cell walls, and quartz sand, which can further abrade the outside of cell. The conserved regions from 16S rRNA and 18S rRNA were selected as target sequences of bacteria and yeast respectively. We evaluated the technique for the detection and identification of which species it was in artificially infected milk and milk from cows with moderate or severe clinical mastitis. The performances of the double-PCR and traditional culture method were evaluated on 84 mastitis milk samples. The results indicated that the sensitivity of double-PCR in detecting bacteria and yeast was 102 CFU/mL and 103 CFU/mL, respectively. There was no significant difference as compared to the culture method in detecting bacteria (P>0.05), but doublePCR was more sensitive than the culture method in detecting yeasts (P<0.01).