The cationic gemini surfactant ethylene bis(1-dodecyldimethylammonium) dibromide was used as a dynamic coating to control EOF and prevent wall adsorption of basic proteins in CE for the first time. This gemini surfactant shows a more powerful capability in EOF reversal than traditional single-chained surfactant. The gemini surfactant reverses the EOF at a concentration level even less than 0.01 mM, and the EOF magnitude is affected by surfactant concentration, pH, ionic strength, and ions added in buffer. Highly efficient and rapid protein separation (N > 300 000) was obtained with buffer containing 2 mM gemini surfactant under pH ranging from 3 to 6. The effects of surfactant and buffer concentration on protein separation were investigated in detail. Under the optimal conditions, good repeatability (RSD of migration time <0.6% for run-to-ran and <2.5% for day-to-day assays) and recovery (>90%) of tested proteins were obtained. This new dynamic coating is also suitable for biosample analysis.