Objective：To introduce a “Four-step method” for extraction and separation of Candida albicans total proteins.Methods: Proteins of C. albicans were extracted step-by-step with 4 kinds of solutions with different solubilities. After quantification,the protein samples were separated by isoelectric focusing electrophoresis and then by SDS-PAGE. Results: Proteins with different solubilities were successfully extracted step-by-step from C. albicans and were separated by two-dimensional gel electrophoresis. Conclusion: The “Four-step method” for extraction of C. albicans proteins is an effective approach to study C. albicans membrane proteome and lays a foundation for further investigation of the mechanisms of antifungal agents and drug resistance in C. albicans.