2-Deoxyribose aldolase-catalyzed CC bond-forming reactions have become 1more and more important in synthesis of statins and other drug intermediates. Many methods have focused on improving the aldolase properties and harvesting new aldolases, but a good outcome depends on the efficiency of the high-throughput screening system. We have developed a visible green fluorescence probe based on a coumarin derivative, which can be reversibly modulated by a retro-aldol reaction catalyzed by 2-deoxyribose aldolase for selecting aldolase mutants with high activity. This assay system provides a convenient and effective way for high-throughput screening aldolases as the green fluorescence is sensitively detected and daylight-viewable without the need for specialist equipment. We used our probe to successfully harvest aldolase mutants with higher activities than the parent from a random mutagenesis library. (C) 2015 Elsevier Inc. All rights reserved.