The invention discloses a preparation method of an optical glucose sensitive film. Glucose oxidase is fixed on silicon oxide mesoporous foam (SiMCF) and silicon oxide nano-spheres (SiNP) which are mixed according to a certain ratio, and polyacrylamide (PAM), cellulose acetate (CA) and polyvinyl alcohol (PVA), which have good biocompatibility, are used as embedding materials; a refractive index type nano glucose sensitive film is prepared on a gold film by adopting a spin-coating method or a Czochralski method to form a glucose sensing probe. The glucose sensing probe is placed in a glucose solution to be detected, and a glucose molecule and dissolved oxygen in the solution enter the glucose sensitive film on the sensing probe and are subjected to oxidization reduction reaction under the catalysis effect of GOD (Glucose Oxidase) so that the refractive index to the sensitive film has monotonous change along the concentration of glucose.