The invention relates to an African swine fever fluorescent PCR (polymerase chain reaction) assay reagent, an African swine fever fluorescent PCR assay kit and application thereof, which belong to the field of bioengineering. With the p72 gene as a reference, a pair of specific PCR primers and a TaqMan fluorescent probe are designed and optimized. Moreover, the primers are designed with the full length of the p72 gene to amplify the p72 gene, and the PCR product is cloned into a pGEM-T vector. Standard curves are drawn with a positive plasmid as a standard product, and the assay range is 10 to 1.0*10<7> copies. In the whole process of assaying African swine fever by an African swine fever fluorescent PCR method disclosed by the invention, only 2.5 to 3 hours are taken from DNA extraction to the appearance of an assay result, manual operation is greatly reduced, and time is greatly shortened. Furthermore, a plurality of samples can be assayed each time, and particularly assay of a large batch of samples can be realized.