The aim of the study is to establish a procedure on root protein extraction applied for proteomics analysis in Brassica napus seedlings.Three methods (TCA/acetone,phenol extraction and phenol extraction-methanol/ammonium acetate precipitation) for protein extraction were assessed with Brassica napus cultivar “Qingyou 10” root at seedling stage as experiment materials.The protein concentration was determined using Bradford protein assay with BSA as a standard,and the protein samples were separated by SDS-PAGE and two-dimensional electrophoresis (2-DE) in turn.Then,the 2-DE gel images were analyzed by PD quest,detecting 644,645,669 clear spots,respectively.Evaluating comprehensively the efficiency of protein extraction among the three methods and optimal 2-DE condition,modified phenol extraction,along with the 2-DE parameters,17 cm (pH 5-8) IPG strip,150 μg protein loading volume and 10% PAGE gel concentration,were chosen to obtain high reproducible and clear 2-DE gel image,which could satisfy the demand for protein isolation and identification in proteomics study.