Bi-functional BaTiO3/CA films were prepared with 500 nm BaTiO3 microspheres and cellulose acetate. SEM, contact angle meter, and UV-visible spectrophotometer were used to characterize them. The effect of BaTiO3 morphology, mass ratio of CA to BaTiO3, mixing time, different surfactants and dosages, solid content and films thickness on morphology, diffusion performance and light reflectivity of bi-functional films were studied. The optimal condition for preparing the bi-functional film were as follows: BaTiO3 was 500 nm microspheres. Mass ratio of CA to BaTiO3 was 0.05. Mixing time was more than 4 h. Surfactants was Triton X-100 which dosages was 20 μL/ 10 g (Triton X-100/ BaTiO3). Solid content was 44.9% and film thickness was 0.4 mm. Bi-functional films prepared in the condition were smooth and thermal stable. The light reflectivity of the bi-functional BaTiO3 film in the 460~800 nm range was higher than 97.5%. Serum diffused in the bi-functional BaTiO3 film reacted in the reagent film and then formed uniform colorful spots. The SD of the diameter of the five parallel samples was 0.055, which showed a good repeatability. The linear range of GLU and URIC reagents prepared with BaTiO3/CA bi-functional films was wider than that prepared with TiO2/CA films.