Titanium-substituted mesoporous molecular sieve SBA-15 have been first successfully synthesized by a new direct approach under conventional hydrothermal conditions. The products obtained by reacting titanium butoxide with acetylacetone were used as the titanium precursor. By means of X-ray diffraction, nitrogen adsorption, UV-Vis diffuse reflectance, a successful isomorphous substitution of titanium in the silica framework of SBA-15 samples with Si/Ti = 50, 25, 20 has been proved, the substitution of Ti for Si does not change the highly ordered two-dimensional hexagonal mesostructure of SBA-15. Titanium is totally tetrahedral and highly dispersed. Higher titanium loading leads to the formation of titanium dioxide. Comparing with two-step preparation method, it shows that under the direct synthesis method, or one-step preparation method, titanium and silicon precursor hydrolyze harmoniously. Ti-SBA-15 demonstrates relatively high catalytic ability on the oxidation of cyclohexene. The differences of structure and catalytic ability between Ti-SBA-15 synthesized by one-step method and two-step method are explained.