The invention discloses primers and a probe composition for detecting an ass-derived component, a horse-derived component and a bovine-derived component in cosmetics, as well as a kit comprising the primers and the probe composition, wherein the primers comprise a pair of universal primers, the probe composition comprises four Taqman probes, and the nucleotide sequences of the primers and the probe composition are shown as SEQ ID NO.1-7. The invention further discloses a multiplex fluorescence PCR detection method for detecting the ass-derived component, the horse-derived component and the bovine-derived component in the cosmetics. The multiplex fluorescence PCR detection method comprises the following steps: taking a to-be-tested sample, extracting the DNA, carrying out PCR amplification on the DNA by utilizing the primers, the probe composition or the kit, collecting fluorescence signals, and determining that whether the to-be-tested sample contains the ass-derived component, the horse-derived component and the bovine-derived component or not according to the fluorescence signals. With the adoption of the detection technology, the false negative result during the PCR reaction can be effectively avoided, and the detection technology has the advantages of being accurate, stable, easy to operate, extremely high in sensitivity, strong in specificity, and the like.