In order to be able to detect the expression of a gene in individual cells, the ability to isolate and lyse a single cell and to perform reverse transcription polymerase chain reaction (RT-PCR) in one device is important. As is common, when performing cell lysis and RT-PCR in the same reaction chamber, it is necessary to add the reagent for RT-PCR after cell lysis. In this study, we propose an original formula for cell lysis and RT-PCR in the same reaction chamber without the addition of reagent by only a heat process, which we termed hot cell-direct RT-PCR. Hot cell-direct RT-PCR was enabled by using Tth DNA polymerase, which is a thermostable polymerase and has high reverse transcription activity in the presence of manganese ions. Direct detection of RT-PCR products was performed by detecting fluorescence with the use of a double-dye fluorescent probe. We attempted to detect the mRNA of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene in isolated Jurkat cells on a microfluidic device, which we had already developed for single cell isolation. After cell isolation and successive hot cell-direct RT-PCR on the device, fluorescent signals from RT-PCR products for a single cell were detected and differentiated from the chamber containing no cells. A highly positive linear relationship (r = 0.9933) was observed between the number of chambers containing cell(s) and those containing RT-PCR products from 10 to 400 cells μL(-1). Thus it was possible to use the novel hot cell-direct RT-PCR method to detect the expressed gene in isolated cells.