Fatty acid esters are widely used in fragrance compounds, solvents, lubricants, and biofuels. Enzymatic synthesis of these esters in aqueous phase is an environmentally friendly approach. In this study, an esterase RasEst3 from Rasamsonia emersonii was identified for fatty acid ester synthesis through sequence alignment. The gene encoding RasEst3 was heterologously expressed in Escherichia coli BL21(DE3), and its enzymatic properties were analyzed. The enzyme exhibited optimal activity at pH 3.5 and 30 °C, with a preference for medium-chain substrates. Structurally, RasEst3 contains a lid domain and a catalytic domain, with a catalytic triad composed of Ser146-His227-Asp214. The smaller pocket spatial site resistance and the hydrophobicity of the substrate channel facilitate effective substrate binding to the active center. Site-directed mutagenesis and molecular dynamics simulations revealed that the oxygen anion holes formed by Gly69 and Thr70, along with the π-bond stacking formed by Tyr112 and Tyr145, play crucial roles in catalysis. After removing a loop region from RasEst3, its ethyl octanoate synthesis activity increased by 253.22 % compared to the wild-type enzyme. This study not only clarifies the structure-function relationship of RasEst3 but also provides valuable insights for developing novel biocatalysts in green chemistry.