A two-dimensional electrophoresis protocol suitable for the separation of proteins from sweet corn leaves was established by using improved TCA/acetone method for total proteins extraction. Two-dimensional electrophoresis conditions such as lysis buffer, protein loading quantities, isoelectric focusing time were modified and improved. The use of modified lysis buffer can lighten the protein horizontal drag; the maximum number of spots were larger than 750, acquired at 500 μg protein loading quantities; the best total isoelectric focusing time was 50 000 Vh; the linear regression correlation coefficient of spots was greater than 0.93, which showed high experiment repeatability.