Alcohol acyltransferases (AATs) enables microbial biosynthesis of a large space of esters by condensing an alcohol and an acyl-CoA. However, substrate promiscuity of AATs prevents microbial biosynthesis of designer esters with high selectivity. Here, we developed a high-throughput microbial screening platform that facilitates rapid identification of AATs for designer ester biosynthesis. First, we established a microplate-based culturing technique with in situ fermentation and extraction of esters. We validated its capability in rapid profiling of the alcohol substrate specificity of 20 chloramphenicol acetyltransferase variants derived from Staphylococcus aureus (CATSa) for microbial biosynthesis of acetate esters with various exogeneous alcohol supply. By coupling the microplate-based culturing technique with a previously established colorimetric assay, we developed a high-throughput microbial screening platform for AATs. We demonstrated that this platform could not only probe the alcohol substrate specificity of both native and engineered AATs but also identify the beneficial mutations in engineered AATs for enhanced ester synthesis. We anticipate the high-throughput microbial screening platform provides a useful tool to identify novel wildtype and engineered AATs that have important roles in nature and industrial biocatalysis for designer bioester production.