Abstract: The mammary gland is a complex organ, and associated with immune factors and secretion that play a vital role in protecting the mammary gland from infection or inflammation. Centrifugation, ultracentrifugation, 2-D clean-up kit and TCA/acetone methods were used to prepare samples and corresponding 2-DE maps were compared by PDQuest7.4 software. The results showed that samples prepared by ultracentrifugation resulted in an efficient concentration, desalting and removing non-protein, its 2-DE map has showed clear spots with less background than the others. Precipitation with 2-D clean-up kit and TCA/acetone were also efficient desalting and removing nonprotein, these gel maps have strong spots, but the protein spots less than that of ultracentrifugation method. The results indicated that ultracentrifugation method was a efficient sample preparation of mammary gland for proteomic analysis.