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. 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, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and 2-DE gels. 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. indicates that kenaf leaf protein spots were distributed primarily 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.