Abstract: The optimal fermentation conditions of fructosyltransferase produced by Aspergillus oryzae was studied by single-factor experiment and uniform design. The optimized conditions was temperature 24℃, rotation rate161r/min, medium volume 50mL/250mL, initial pH5.0 and inoculum size 1%. The highest enzyme activity was33.07U/mL after 96h, 64.6% higher than before. The fructosyltransferase was separated and purified by using a procedure including column chromatography separation by Desalting, DEAE FF and SepHacryl S-100 HR. The final purified fold, specific activity and yield of the purified enzyme were 29.35, 231.14U/mg and 48.9%, respectively. The purified enzyme was confirmed to be homogeneous by SDS-PAGE. Detected by thin layer chromatography ( TLC) , the enzyme was able to generate sucrose- 6- acetate ( s- 6- a) , as proof of fructosyltransferase. Its molecular weight was estimated to be 92.8ku.