Palladium nanoparticles (Pd NPs) are the most widely used functional materials for a variety of catalytic reactions. In this work, we synthesized the size-controlled Pd NPs using amyloid fibrils as bio-templates. First, 1mg/ml insulin peptides were incubated at 80°Cfor 3 days to form protein fibrils. Then, layer-by-layer technology was used to prepare Pd-insulin fibrils multilayer film as catalyst by alternatively depositing insulin fibrils and palladium chloride which has been aged at several different temperatures for two days. The chemical compositions of Pd-insulin film were also characterized by X-ray photoelectron spectroscopy (XPS). The Pd-insulin film demonstrated high isolated yield in promoting Suzuki cross-coupling reaction. In addition, the effect of Pd NPs size on the catalytic activity was also discussed.