Transparent protein film of iron-free cytochrome c (Cyt. c) was successfully manufactured by electrospray deposition (ES) method and the properties of the protein film were investigated. Excursion temperature dependence of spectral hole profiles in the photochemical hole burning for the iron-free Cyt. c in protein film and in glassy solution was investigated and compared with that of iron-free porphyrin embedded in a synthetic polymer film to clarify photochemical properties of electrospray deposited protein film. The spectral holes of iron-free porphyrin were thermally more stable in Cyt. c than in polymer, indicating compact packing of the chromophore in the protein. The ES-deposited iron-free Cyt. c in protein film showed less stable spectral hole than that in glassy solution. This difference is attributable to the electrospray deposition process of the protein involving ionization and to subsequent cross-linking of protein molecules.