—We report a new microfluidic biosensor to detect changes in the transcriptional activity of the single genetically modified cells of the Helicobacter pylori bacterium in response to exogenous factors. A microfluidic chip was used as the basis for the development of the biosensor, in which the channel wall surface was modified to provide effective cell immobilization and maintenance of cell viability. In order to demonstrate the efficiency of the biosensor, the changes in the fluorescence of the GFP protein, the gene of which was under the control of the pH-dependent virulence genes of H. pylori, in response to acidic stress, were registered. The developed microfluidic biosensor may be used not only for the basic microbiological research, but also for the diagnostics of different pathological states.