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To establish a suitable and effective protocol of protein extraction for two-dimensional gel electrophoresis (2-DE) analysis in kenaf leaf tissues,three extraction methods (trichloroacetic acid/acetone,urea/thiourea,and phenol extraction methods) were applied to the extraction of kenaf leaf protein.The results were compared in regard to protein extraction efficiency,sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),and 2-DE gels.Furthermore,the 2-DE system was optimized for four aspects:the pH range of IPG (immobilized pH gradient) stripes,sampling methods,sample volumes,and concentration of polyacrylamide gels.The data presented showed that the phenol extraction method is the best method to perform 2-DE analysis of kenaf leaf protein.The protein extracted from phenol extraction method reached the purity of (26.40±0.859)％,showed (25.67±1.53) protein bands in one dimension SDS-PAGE gels,and (1 374±54.44) protein spots on 2-DE gels.The research also indicates that kenaf leaf protein spots were distributed mainly within the pH range of 4-8.More clear background with a better distribution effect and many protein spots could be obtained on 2-DE gels under the conditions of active rehydration loading,24 cm IPG strips (linear pH gradient of 4-7),1.4 mg samples,and 12％ SDS-PAGE gels.