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Porous titania beads were synthesized by a sol−gel templating method using agarose gel as template. The morphology and structure of the materials were characterized by X-ray diffraction, scanning electron microscopy, high-resolution transmission electron microscopy, and N2 adsorption analysis, etc. It was found that the size and pore structure of the resultant titania beads could be readily controlled by using various sized templates and by changing the precipitation conditions. Under optimized conditions (i.e., impregnating agarose gel into titanium precursors for 6 h, three cycles of repeated impregnation and hydrolyzing process, and calcination at 450 °C), porous titania beads of 7−150 μm in mean diameters were fabricated by using different sized agarose beads as templates. The beads displayed perfect spherical shape, with an average pore size of 6 nm and specific surface area of 69.9 m2 g−1. The titania beads of 15 μm in average diameter were further characterized for application as liquid chromatographic packings. Flow hydrodynamic experiment indicated that they possessed high mechanical strength to withstand a pressure drop up to 12 MPa. The column efficiency reached 6165 plates m−1 for acetone and 4650 plates m−1 for N,N-dimethylaniline under nonretained condition. Moreover, baseline separation of three aniline derivatives featuring only small structural differences was realized by using this packed column.