The goal of this work was to improve the bioluminescence-based signaling assay system to create a practical application of a biomimetic odor sensor using an engineered yeast-expressing olfactory receptors (ORs). Using the yeast endogenous pheromone receptor (Ste2p) as a model GPCR, we determined the suitable promoters for the firefly luciferase (luc) reporter and GPCR genes. Additionally, we deleted some genes to further improve the sensitivity of the luc reporter assay. By replacing the endogenous yeast G-protein α-subunit (Gpa1p) with the olfactory-specific Gαolf, the optimized yeast strain successfully transduced signal through both OR and yeast Ste2p. Our results will assist the development of a bioluminescence-based odor-sensing system using OR-expressing yeast. Biotechnol. Bioeng. 2012; 109: 3143–3151. © 2012 Wiley Periodicals, Inc.