A fast method for the preparation of cytochrome C (Cyt-C) of high purity from pig heart is presented. A simple-type column of hydrophobic interaction chromatography packed with big particles was employed for the fast purification in laboratory scale. The purity of the purified Cyt-C was verified by reversed-phase liquid chromatography, UV spectroscopy, and iron content in it. Compared with the common method, the new method has advantages of higher speed, simpler purification processes, higher mass recoveries and higher purity.