Objective To establish a rapid monitoring method of the three common bacteria in mice frozen resources, such as embryo, sperm, etc. Methods To extract DNA of the three positive bacteria(Staphylococcus auerus, Klebsiella-pneumoniae and β-hemolyticstreptococcus), and establish PCR monitoring method of the three positive strains through designing primer and refining PCR condition. Then extract total DNA of the frozen resources, detect the DNA according to the PCR condition of the three positive bacteria, some samples were detect by fluorescence quantitative PCR at the same time. Results ①successfully establish a PCR detection method of the three positive bacteria, the minimum detectable concentration of Staphylococcus auerus, Klebsiella pneumoniae and β-hemolytic streptococcus is 4.19×10^-5 ng/μL, 1.98×10^-5 ng/μL and 1.07×10^-3 ng/μL.②Proved that the three bacteria doesn't exist in the sample by normal PCR and fluorescence quantitative PCR methods. Conclusions Establising a rapid monitoring method of common pathogens of frozen embryo and sperm in mice.