Proteomics has become a powerful technology being successfully used in plant science research to investigate different biological processes, including plant genetics, development, physiological ecology and responses to climate change. Two-dimensional electrophoresis (2-DE) is a potential analytical tool in proteomics to identify qualitative and quantitative changes of proteins and reveal the changes of protein expression under different growth conditions. However, protein sample preparation is the key fundamental steps of 2-DE analysis, and it is also a prerequisite for proteomics analysis. L638-y is a chlorophyll-deficient mutant naturally generated from the wild type L638-g in Brassica juncea L., which is typical characterized with yellow leaves and no other different traits from the wild type except leaves color. In order to efficiently analyze the differential expression proteins between the mutant L638-y and its wild type L638-g, two protein extraction methods for proteomic analysis, TCA/acetone extraction method and an improved polyethylene glycol ( PEG) fractionation method, were compared. Total proteins were extracted from leaves of the mutant L638-y and its wild type L638-g at five leaves period with the two protocols, then the samples were analyzed by 2-DE, pH of IPG strip and concentration of SDS-PAGE being optimized. The results showed that when using 17 cm linear IPG strips pH ranged from 4 to 7, 11% SDS-PAGE, loading 180 μg/350 μl, proteins were better separated. Much more protein spots had been observed in 2-DE maps of the protein samples prepared by the PEG fractionation than TCA/acetone precipitation. In 2-DE profiles of the protein sample from mutant L638-y prepared by the PEG fractionation, as much as 1 235±6 protein spots were identified, 330 more spots than those by TCA/acetone precipitation method. By using the PEG fractionation, 190 differential protein spots were identified between the 2-DE maps of the mutant L638-y and wild type L638-g, 100 more spots than those by TCA/acetone precipitation. Thus, the improved PEG fractionation is more efficient and simple for proteomic analysis of chlorophyll-deficient mutants in Brassica Juncea L.