The TS-1/diatomite catalyst was prepared for the hydroxylation of phenol with H2O2 in the fixed-bed reactor and the effects of pretreatment on the properties of TS-1/diatomite were studied by FT-IR, XRD, UV-vis, ICP-AES, BET surface area and NH3-TPD techniques. It is shown when the catalyst is pretreated by the KAc, NaAc, NH4Ac, NH4Cl or HNO3 aqueous solution, the framework structure of TS-1 is not destroyed and titanium in the framework is not removed. The surface area of catalyst has no obvious change compared with that of the untreated catalyst. But the extra-framework TiO2 can be removed partly, which leads to the slight increase of the crystallinity of catalyst and the decrease of acid concentration on the surface of the TS-1/diatomite catalyst. As a result, the activity, selectivity and utilization of H2O2 for hydroxylation of phenol are improved. After the TS-1/diatomite catalyst is pretreated by the NH3•H2O, Na2CO3 or Na3PO4 solution, its framework silicon is dissolved partly in the base solution and the framework structure of TS-1 is destroyed. While the crystallinity and surface area of catalyst decrease and the concentration of acid sites on the surface of catalyst increased slightly. As a result, the catalytic activity of the TS-1/diatomite catalyst for hydroxylation of phenol descended or deactivated completely.