The invention relates to a primer probe composition capable of detecting five pathogenic bacteria at the same time. The primer probe composition comprises a sequence primer sequence or a probe sequence shown as SEQ ID No.1-15 as shown in a chart 1; a report radical group is modified at the 5' end of each probe sequence, and a quenching radial group is modified at the 3' end of each probe sequence; the report radial group has FAM, FITC, HEX, JOE and CY3, and the quenching radial group has TAMRA, BHQ and Eclipse. According to a detection method of five-fold real-time fluorescence quantification PCR, the five pathogenic bacteria such as salmonella, vibrio vulnificus, Shigella, staphylococcus aureus and Listeria monocytogenes can be detected at the same time in the same reaction system. Moreover, the five pathogenic bacteria are detected by using the five-fold real-time fluorescence quantification PCR method, the sensitivity and specificity of detection can be improved, the detection efficiency can be greatly improved, step complication and long detection period existing in conventional detection are remedied, and the primer probe composition cannot be applicable to the defects of large-scale quick detection.