The invention discloses a tobacco acyl sugar-acyl transferase gene NtASAT3 and an encoded protein and application in sucrose ester biosynthesis of the gene. The synthesis of sucrose triester can be regulated by the encoded protein of the gene in a safer ammonium acetate buffer solution. The entire sucrose ester biocatalysis reaction is carried out in the ammonium acetate buffer solution, and no other substances are added except sucrose, acyl-COA and catalytic NtASAT1-NtASAT3 proteins. Compared with the previous sucrose ester synthesis methods, in which toxic reagents are required as substratesor solvents, the entire catalytic reaction system has high safety, simple reaction substrates and relatively single system products, and all the products are sucrose esters, which are liable to subsequently separate and utilize. Therefore, the NtASAT3 gene and the protein of the gene have good application value and prospects in sucrose ester biosynthesis.