Abstract: To establish an appropriate two- dimensional electrophoresis system of melon fruit, the variety of Cucumis melo‘Jingmi 11’was used as material, parameters including protein sample preparation, isoelectric focusing condition and the pH gradient of IPG strip were optimized. The result showed that the optimized system included the following steps: extracting the total protein from melon fruit by phenol- methanol/ ammonium acetate, separating the proteins with 24 cm pH 4-7 IPG strips, loading protein samples of 800μg, 120000 Vhs isoelectric focusing and staining the gels by colloidal coomassie brilliant blue staining after SDSPAGE electrophoresis. Clear background, reproducible profiles with high resolution were obtained by this optimized 2-DE system of melon fruit.