The invention discloses a method for detecting real-time fluorescence quantitative RT-PCR of a single cell of foot and mouth disease virus genome RNA. The method utilizes a microinjection instrument to separate single cell of a foot and mouth disease virus; and after cracking, the fluorescence quantitative RT-PCR is used to carry out quantitative analysis. Visible operation of the microinjection instrument can rapidly and accurately separate out the single cell; and the microinjection instrument is combined with the high-sensitivity fluorescence quantitative RT-PCR to realize the detection of the quantity of the virus genome RNA in the single cell. The method can be used for researching virus copying on the level of the single cell and the relation between the single cell and a host cell and provides a new method for in-depth research of virus infection cytobiology. The technology for separating and cracking the cells has wide applicability, can be directly applied to the separating pretreatment of other kinds of virus-infected cells or normal cells in order that the method can be also used for the detection of other RNA virus genomes, the copying of normal cell genomes and the research on a transcribed molecular mechanism.