Staphylococcus spp., is a common bacteria in cattle mastitis. Detection of the low number of bacteria requires a sensitive method. We used multiplex polymerase chain reaction (m-PCR) assay to detect the genes encoding SEA, SEB, SEC toxins, that are specific in S. aureus bacteria. The gene of 23S rRNA that is conserved in all Staphylococcus spp., also was used. Due to the stiffness and low effective enzyme activity such as lyzozyme, proteinase k, and mutilysine on bacterial cell wall, it was lysed with liquid nitrogen, and multiplex PCR was performed after DNA extraction. The obtained results in molecular method were compared with traditional cell culture. The total number of 48 samples of non pasteurized milk was collected in Tehran province restricts from dairy cattle housing, and 4 positive samples were detected by PCR and microbial cell culture methods. The result of antibiogram showed that Ciprofloxacin, Gentamycin and Rephampine are the best choice of antibiotic for treatment of Staphylococcus spp., mastitis infectious. The accuracy of the test was monitored by using O/N cell culture serial dilution (1 to l06) of Staphylococcus spp., bacteria (OD600=0.02:107 cell). It showed that the sensitivity of PCR is 10 bacteria per ml of cells within few hours, meanwhile in bacterial blood agar cell culture; the number of 100 cells was detected after 48 hours. The result indicates that a molecular technique is more sensitive and rapid compared to traditional bacterial culture method.