The aims were to optimize the extraction methods of Fusarium oxysporum f. sp. Cubense intracellular proteins, and establish the two-dimensional electrophoresis system for intracellular proteins. The extraction methods of intracellular proteins were optimize based on the past reports, proteins for two-dimensional electrophoresis were extracted, and mass spectrum was used to identify the proteins for verify the mass spectrum compatibility. The sufficient mass was obtained through the expansion culture methods, and the bacteria were collected with filtration for protein extraction. Vortex 30 min could damage the cell effectively; the efficiency of protein extraction was improved. The protein was washed twice by methanol and acetone to remove protein impurities effectively. The high quality 2-DE profiles were obtained. 10 protein spots were selected and identified by MS, successfully obtained 8 protein spots which represented 8 unique proteins. These results suggested that the extracted proteins and method of in-gel digestion had good MS compatibility, and could also provide foundation for further investigating the differentially expressed proteins between the difference thalli.