Staphylococcus aureus, Escherichia coli and Bacillus cereus are the major agents of cow endometritis in dairy cows. A multiplex PCR (SEB-mPCR) was established based on the conserved genes of S. aureus, E. coli and B. cereus, and the detection limits were 103, 102 and 103 CFU mL-1, respectively. SEB-mPCR could not amplify genomic DNA of pathogenic bacteria of other common bovine diseases. A total of 309 vaginal discharge samples from cows with endometritis were tested by SEB-mPCR. Of the samples, 23.95% had the three kinds of bacteria detected, 17.15% had S. aureu and E. coli, 9.39% had E. coli and B. cereus, and 9.71% had S. aureus and B. cereus. The rates of infections with S. aureus, E. coli and B. cereus were 11.35, 16.18 and 9.06%, respectively. Therefore, SEB-mPCR has a potential as a diagnosis tool for endometritis in dairy cows.