Abstract: [Objective and Method]The objective of this paper was to compare with three different protein extraction methods of Gossypium hirsutum, cv. Xuzhou 142 and the effects of various lysis buffers on thedouble direction electrophoresis (2-D). The loading quantity of sample and staining method were also optimized for the 2-D analysis.[Result]The results showed that the protein content of cotton fibre extracted by saturated powder was highter than that extraced by other methods and the electrophoresis strip of SDS-PAGE were clear;the solubility raised whose protein were dissolved in the mixture of thiourea and CHAPS; the more suitable loading quantity was 300 μg to the pH 3-10 7 cm IPG strip. Furthermore, the protein spots were increased and the background was clear by the modified Coomassie Brilliant Blue method.[Conclusion]The double direction Gel Electrophoresis method used to extract cotton fibre protein was initially established.