Abstract: [Objective ] To develop a multiplex fluorescent PCR assay method to detect swine-derived and bovine-derived materials in animal products. [ Method ] Two pairs of specific primers and two fluorescent-labled probes were designed and synthesized according to mitochondrial DNA gene sequences. The multiplex fluorescent PCR reaction system was established through the optimized screening of reaction system and reaction conditions, and swine-derived and bovine-derived materials in animal products were detected in the same fluorescent PCR reaction system. Finally, the sensitivity and specificity of the assay were evaluated. [ Result ] The method successfully detected swine-derived and bovine-derived materials from 16 animals. Swine-derived and bovine-derived materials could obtain responsible S-type amplification curve, while there was no amplification curve from the other 14 animals. The detection limits of the assay for swine and bovine were either up to 10^-5, which was consistent with that of single-weight fluorescent PCR assay method. [Conclusion ] The multiplex fluorescent PCR developed in the study has strong specificity and good sensitivity, so it is suitable for the detection of swine-derived and bovine-derived materials in feed, meat products, milk products, etc..