Here, we describe a yeast-based fluorescence reporter assay for G protein-coupled receptor (GPCR) signalling using a flow cytometer (FCM). The enhanced green fluorescent protein (EGFP) gene was integrated into the FUS1 locus as a reporter gene. The engineered yeast was able to express the EGFP in response to ligand stimulation. Gene-disrupted yeast strains were constructed to evaluate the suitability of the yeast-based fluorescence screening system for heterologous GPCR. When receptor was expressed by episomal plasmid, the proportion of the signalling-activated cells in response to ligand stimulation decreased significantly. The GPCR-signalling-activated and non-activated cell clusters were individually isolated by analysing the fluorescence intensity at the single-cell level with FCM, and it was found that the plasmid retention rate decays markedly in the non-activated cell cluster. We attributed the loss of plasmid to G1 arrest in response to signalling, and successfully improved the plasmid retention rate by disrupting the FAR1 gene and avoiding cell cycle arrest. Our system will be a powerful tool for the quantitative and high-throughput GPCR screening of yeast-based combinatorial libraries using FCM.