Background. Sub-cellular compartmentalization is used by cells to create favorable microenvironments for various metabolic reactions. These compartments concentrate enzymes, separate competing metabolic reactions, and isolate toxic intermediates. Such advantages have been recently harnessed by metabolic engineers to improve the production of various high-value chemicals via compartmentalized metabolic engineering. However, measuring sub-cellular concentrations of key metabolites represents a grand challenge for compartmentalized metabolic engineering. Methods. To this end, we developed a synthetic biosensor to measure a key metabolite, acetyl-CoA, in a representative compartment of yeast, the peroxisome. This synthetic biosensor uses enzyme re-localization via PTS1 signal peptides to construct a metabolic pathway in the peroxisome which converts acetyl-CoA to polyhydroxybutyrate (PHB) via three enzymes. The PHB is then quantified by HPLC. Results. The biosensor demonstrated the difference in relative peroxisomal acetyl-CoA availability under various culture conditions and was also applied to screening a library of single knockout yeast mutants. The screening identified several mutants with drastically reduced peroxisomal acetyl-CoA and one with potentially increased levels. We expect our synthetic biosensors can be widely used to investigate sub-cellular metabolism and facilitate the "design-build-test" cycle of compartmentalized metabolic engineering.