In order to establish a suitable and effective protocol for two-dimensional gel electrophoresis (2-DE) analysis of the buds of Brassica rapa L. ssp. Pekinensis, whole proteins from fertile buds of the A/B line AB01 were extracted using three different methods (Tris-acetone-phenol method, Trizol precipitation method, TCA-acetone precipitation method). Furthermore, four aspects of the 2-DE system were optimized, including the IEF protocol, SDS-PAGE gel concentration, pH range of IPG strips and protein sample volume. The results show that the TCA-acetone precipitation method is the most effective method for whole protein extraction, and an optimized 2-DE system using the 20,000 Vh IEF protocol, pH 4-7 IPG strips, 12% SDS-PAGE gel concentration and protein sample volumes of 250 μg is suitable for 2-DE analysis of fertile bud protein in Chinese cabbage. These results can be used to facilitate proteomic studies of genic male sterility in Brassica rapa L. ssp. Pekinensis.