Foot-and-mouth disease virus is a positive-sense, single-stranded RNA virus with a negative strand as its replication intermediate, which can cause severe acute infection in sensitive cell lines. To investigate better the actual state of virus infection, there is a need to measure the amount of FMDV RNA in a single acutely infected cell rather than in a large number of cells. Therefore, in the present study, a strand-specific single-cell quantitative real-time RT-PCR was developed to analyze the RNA or FMDV. This new method uses two techniques in concert with each other: a technique for isolating single cells with micromanipulators, which is coupled to an assay for detecting viral RNA by real-time RT-PCR. In the assay of acute infection, 185 of 224 (82.6%) single-cell samples were positive and contained viral genome copies ranging from several to thousands, and up to 1,000,000 copies. However, not all cells were infected and there were differences in the number of viral RNA copies between cells. A single-cell quantitative RT-PCR was validated to be feasible and effective.