TThe active sites of β-fructosyl-transferase from Aspergillus oryzae GX0011 were investigated by means of chemical modification and kinetic study. NBS(N-bromosuccinimide)、PMSF(Phenylmethyl-sulphonyl fluoride)、EDC(Carbodimide) seriously inactivated the enzyme but not in the presence of substrate. The inactivation reactions followed pseudo-first-order kinetics. Further kinetic analysis of the inactivation indicated that at least one serine/threonine residue, one tryptophan residue and one asparic acid/glutamic acid residue might be involved in the active site of the enzyme. PCMB(p-chloromercuribenzoate) and TNBS(2,4,6-trinitrobenzene sulfonate) could inactivate the enzyme even in the presence of substrate. This implied that cysteine and lysine residues might not be in the active site of the enzyme but involved in maintaining the conformation of the enzyme. DEPC(Diethylpyrocarbonate)、AA(acetylacetone) and NAI (N-acetyl-imidazole), did not obviously influence activity, thus eliminating the possibility that histidine, arginine and tyrosine participated in catalysis.