Protein thin film (mainly silk fibroin) was prepared by pulsed laser deposition (PLD) with 1064nm IR-beam and via colloid chemical routes. Thickness, surface roughness, and microstructures of the deposited film were examined by quartz crystal microbalance sensor, field emission scanning electron microscope (FE-SEM), and atomic force microscope (AFM). The laser power density was varied systematically for PLD to control the microstructures of the film and the secondary structure (β-sheet, α-helix, or random coil) of the protein. Secondary structure of the target and film was examined by FT-IR. Films prepared by PLD comprise by agglomerated particles with their primary particle size around 30nm. The size of the primary particles was uniform, especially for the film prepared at low laser power density. At low laser power density, proportion of β-sheet increased and that of random coil decreased. Proportion of random coil was also increased by the wet colloidal process. PLD with low power density is most suitable to preserve the secondary structure in the protein thin film.