The invention relates to a titanium dioxide microsphere for high performance liquid chromatograph (HPLC) and a preparing method thereof. The titanium dioxide microsphere has a mesopore structure, the particle diameter (diameter) is 0.5-5 mum, and the specific surface area of the pore diameter is 100m<2>/g-130 m<2>/g. The titanium dioxide microsphere is prepared by an improved sol-gel and water thermal crystallisation method. The prepared titanium dioxide microsphere has the advantages of monodisperse property, mesopore structure, uniform particle size, conformance of the surface structure (large pore diameter and large specific surface area) to the chromatograph requirements, no need of grading treatment, and the like. In addition, in the process of the preparation method, the particle diameter of the object (titanium dioxide microspheres) can be controlled (i.e. the microspheres with different particle diameters can be easily obtained through adjusting different titanate concentrations), and the whole preparation process is simple and easy.