The invention discloses a one-step multiplex real-time fluorescence quantitative PCR detection primer and probe for SVA, FMDV, SVDV and VSV, and belongs to the field of biotechnology detection. A kitused for simultaneously detecting the SVA, FMDV, SVDV and VSV is developed based on the provided primer and probe. The provided kit and a detection method thereof have the advantages of simple and convenient operation, high specificity, high sensitivity and good repeatability, can achieve simultaneous and accurate qualitative and quantitative detection of the SVA, FMDV, SVDV and VSV, can play an important role in the detection of the SVA, FMDV, SVDV and VSV and vaccine production, such as quality monitoring and rationalized vaccine distribution during the vaccine production, and have broad application prospects.