The charge trapping of the cytochrome c was investigated by electrostatic force microscopy (EFM). The cytochrome c was modified by SPDP and deposited onto the gold substrate by self-assembly technique. The charge was transported to heme groups of cytochrome c by scanning tunneling microscopy (STM) tip, and it was verified that the charge was trapped onto the cytochrome c film surface by EFM. The charge distribution on the cytochrome c surface was observed by EFM measurement.