Barium titanate/cellulose acetate (BaTiO3/CA) membranes with reflection and diffusion functions were prepared with 500 nm BaTiO3 microspheres as raw material and cellulose acetate as binder. SEM, contact angle meter, and UV-visible spectrophotometer were used to characterize the samples. The effects of BaTiO3 morphology, mass ratio of CA to BaTiO3, stirring time, surfactant type and dosage, solid content and wet film thickness on the morphology and diffusion properties of bi-functional membranes were studied. The results showed that the optimal conditions for preparing BaTiO3/CA membranes were as follows: BaTiO3 microspheres particle size 500 nm, m(CA):m(BaTiO3)=0.050:1, stirring time more than 4.0 h, surfactant being Triton X-100, Triton X-100/BaTiO3 20 μL/10 g, acetone 15 mL, solid content 44.9%, and wet film thickness 0.40 mm. Under these conditions, the prepared membranes were flat and smooth with good thermal stablity. After the serum diffused by the bi-functional membrane to the self-made reagent layer, the spots formed by the reaction were uniform in color. The standard deviation (SD) of the reaction spot diameter of five parallel samples was 0.055, which showed that the diffusion was uniform and has good repeatability. The light reflectivity of BaTiO3/CA membranes in the range of 460~800 nm was over 97.5%. Glucose (GLU) and uric acid (URIC) reagents prepared with BaTiO3/CA bi-functional membranes exhibited wider linear ranges than those prepared with TiO2/CA membranes, which was easier to use. © 2020, Editorial Office of FINE CHEMICALS. All right reserved.