We demonstrated the non‐biofouling performances and binding capabilities of amylose film coated on a glass surface using fluorescence analysis. The amylose film was simply prepared using a drop‐casting method and then functionalized with amine‐terminated biotin through surface organic reactions such as periodate oxidation, amide bond formation, and reduction. The biotin, spatially patterned on the film, selectively interacted with streptavidin labeled with a dye, which enabled the evaluation of the binding capabilities and non‐biofouling efficacy of 0.2 and 0.4 wt% amylose film. These films exhibited similar results, such as excellent non‐biofouling properties and slightly low binding capabilities (minimum concentration of biotin: 10 μM).