In order to establish a twodimensional gel electrophoresis (2DE)protocol for proteomic study of desert chlorella (Chlorella sp. TLD6B), we compared the performances of protein extraction methods from desert chlorella betweenTCA/ acetone precipitation method and Trisol extraction. The chose pH gradient gel strips and protein loading volume were also optimized. Our results showed that: (1) trisol extraction could obtain high pure protein. The improved 2DE system for desert chlorella was come out as follows: we distinguished 726 proteins with linear IPG strips of 24 cm in length with the pH ranges from 3 to 10, 500 μg loading volume, 80000 Vh of IEF time. We distinguished 1230 proteins with linear IPG strips of 24 cm in length with the pH ranges from 4 to 7, 1 000 μg loading volume, 80 000 Vh of IEF time. (2) Ten randomly selected protein spots were identified by using MALDITOF/TOFMS to confirm the establishment of twodimensional electrophoresis protocol could be used for primary analysis of desert chlorella proteome.