The invention relates to a multiplex amplification internal standard PCR (polymerase chain reaction) kit capable of simultaneously detecting Vibrio parahaemolyticus, Escherichia coli O157:H7, Salmonella and Shigella. The kit comprises a PCR reaction premix solution and a reference substance. The detection method comprises the following steps: acquiring a sample, carrying out amplification culture, extracting DNA (deoxyribonucleic acid), preparing a PCR reaction tube, putting the PCR reaction tube on a PCR instrument, carrying out PCR amplification, and carrying out detection result judgment on the PCR amplification product. The kit can simultaneously and quickly detect the four bacteria by one reaction, and can avoid the misjudgment caused by the false negative result due to the inhibiting factor for the DNA polymerase in the sample treatment process. Compared with the existing detection methods, the method provided by the invention is convenient to use, has the advantages of high reliability, short detection period, high sensitivity, high specificity, low cost and simple operation steps, is suitable for high-flux operation and standard operation, and is beneficial to enhancing the food safety detection level and preventing and controlling the foodborne diseases.