The purpose of this research was to establish an optimized system to analyze Apostichopus japonicus proteomics. The protein extraction method, loading methods, sample volume and focusing condition in two-dimensional gel electrophoresis (2-DE) were optimized to obtain best results. Several methods of protein preparation (TCA-acetone precipitation treatment, Tris-HCl treatment, Tris-HCl acetone treatment and pure water treatment) were applied to extract total protein from A. japonicus intestine. The most effective profiles with the most electrophoretic protein spots were obtained by the TCA-acetone precipitation treatment. The 2-DE electrophoretic analysis in intestine achieved an optimized map by loading without buffer solution treatment at the sample volume of 25 μg， The pH of ampholytes range from 4 to 6.5. The samples were electrophoresed for 1000 V and 3 hours， and the gels were immersed in 2.5 ml equilibration buffer and equilibrated for 30 min. The system optimization of protein two-dimensional electrophoresis would provide supports in sea cucumber proteomics study.