AbstractEsters are widely used in food, energy, spices, chemical industry, etc., becoming an indispensable part of life. However, their production heavily relies on the fossil energy industry, which presents significant challenges associated with energy shortages and environmental pollution. Consequently, there is an urgent need to identify alternative green methods for ester production. One promising solution is biosynthesis, which offers sustainable and environmentally friendly processes. In ester biosynthesis, alcohol acyltransferases (AATs) catalyze the condensation of acyl-CoAs and alcohols to form esters, enabling the biosynthesis of nearly 100 different kinds of esters, such as ethyl acetate, hexyl acetate, ethyl crotonate, isoamyl acetate, and butyl butyrate. However, low catalytic efficiency and low selectivity of AATs represent the major bottlenecks for the biosynthesis of certain specific esters, which should be addressed with protein molecular engineering approaches before practical biotechnological applications. This review provides an overview of AAT enzymes, including their sequences, structures, active sites, catalytic mechanisms, and metabolic engineering applications. Furthermore, considering the critical role of AATs in determining the final ester products, the current research progresses of AAT modification using protein molecular engineering are also discussed. This review summarized the major challenges and prospects of AAT enzymes in ester biosynthesis.