Single-cell measurements are necessary for investigating the stochasticity of gene expression because cell-to-cell variation cannot be quantified using population measurements. Here, we incorporated the receding meniscus induced docking method into high-throughput automated fluorescent microscopy for analyzing stochastic nature of the MAPK signaling pathways in the budding yeast, Saccharomyces cerevisiae. Using the technique, we determined the real-time gene expression patterns of the mating responses at single-cell resolution. We also observed that the mating MAPK signaling showed a non-uniform, fluctuating flux in the population of yeast cells analyzed.