The invention discloses a tobacco acylglycosyltransferase gene NtASAT1, protein encoded by the tobacco acylglycosyltransferase gene NtASAT1 and application of the protein in biosynthesis of sucrose ester, and the protein encoded by the tobacco acylglycosyltransferase gene NtASAT1 can regulate synthesis of sucrose monoester in a safer ammonium acetate buffer solution. A whole sucrose ester biological catalytic reaction is carried out in the ammonium acetate buffer solution, and except for sucrose, acyl-COA and catalytic NtASAT1, no other substances are added. Compared with most existing sucroseester synthesis methods in which toxic reagents are used as substrates or solvents, the whole catalytic reaction system has high safety, reaction substrates are simple, the system products are relatively single, all products are sucrose esters, and following separation and utilization are facilitated.