In this paper, the homogeneous diameter TiO2 microspheres were prepared by precipitation method. The structures and characterization of TiO2 microspheres were characterized by XRD, SEM, TG and reflectance spectroscopy. The factors of urea dosage, solvent types and dosage, titanium ion concentration, hydroxypropyl cellulose(HPC) concentration and synthesis time were investigated. The prepared conditions were: c(TiCl4)=0.2 mol/L，V(water):V(n-propanol)=1:3，c(TiCl4)=0.2 mol/L，n(Urea):n(TiCl4)=6:1，HPC concentration was 0.85 g/L, incubated 80 minutes at 90°C and calcinated at 500°C. The yield of TiO2 microspheres was 87.7%.The monodisperse particles were TiO2 and the diameter could reach 2~3 μm with a excellent uniformity which were characterized by XRD and SEM. Furthermore, the reflecting/spreading bifunctional layers prepared with the TiO2 microspheres were studied. The results showed that the reflectance of the bifunctional layer was greater than 90.7% in the range of 400~780 nm. It formed a uniform colour spot after serum spread through bifunctional layer and reacted in reagent layer, which indicated a homodisperse of serum and an excellent reproducibility of this bifunctional layer.