Abstract: A two-dimensional electrophoresis system for total protein from sweet pepper(cv. ‘Hongfang') fruits was established,including optimal conditions of total protein extraction method,pH value of the extract buffer,pH range of IPG strips,loading amount and isoelectric focusing procedure. The results showed that the best image of picture as indicated by protein spot uniformity and background clearness as well as protein point resolution was obtained at pH7.8 for extract buffer and by adopting phenol extraction method,and loading1200μg protein to 17 cm IPG strip of pH5 ~8 and isoelectric focusing at 66000 VH followed by SDS-PAGE,and finally detecting proteins with coomassie brilliant blue G-250 staining. The established two-dimensional electrophoresis system could meet the needs of the subsequent mass spectrometry analysis. The total protein spots in the image of pictures were around 703±17.