Abstract: Conopeptides is one kind of small precious nerve toxin peptides, which is excreted from Conus snails. Because the post translational processing of conopeptides is complicated, it is very difficult to synthesize conopeptides peptide with biological activity by the method named chemical synthesis. Many proteins from Conus snails, such as protein disulfide isomerase (PDI), prolylhydroxylase, proline isomerase, and so on, which promote the synthesis and activity formation of conopeptides. The protein disulfide isomerase (PDI) is an important foldase in folding of the nascent polypeptide chain of the endoplasmic reticulum (ER). The venom of tropic officinal marine cone snails is abundant with PDI which is important in assisting the folding and maturation of secretory conopeptides in vivo. As an effective and direct approach of research in all proteins, proteomics represents a novel methodological way to investigate the expression of all proteins of a cell, body fluid or organism, as well as to analyze their structure, function, interaction and modification. Proteomics has been widely applied in studying regulation of metabolism, genetics and development, physiology and biochemistry, pharmacology and toxicology. And many technologies of proteomics were available to research Conus snails venom protein in integer protein level. The report was to analyze the total proteins by the widely use technologies including two-dimensional electrophoresis (2-DE), mass spectrum, chromatogram analysis and biology informations, so as to lay a good foundation for further research on Proteomics of Conus venom. Previous trials on different electrophoresis methods showed that SDS-PAGE electrophoresis is difficult to detect protein with low molecular weight, while Tricine-SDS-PAGE electrophoresis can detect low molecular weight protein clearly. But in the 2-DE experiment, the Tricine-SDS-PAGE electrophoresis makes protein point deform and gets a blurry 2-DE map for taking more time than SDS-PAGE electrophoresis. So in this 2-DE study of Conus snails venom protein, we use the concentration of 15% SDS-PAGE gel to prepare the second PAGE gel. Such as parameters of sample preparation, isoelectric focusing and gel electrophoresis were explored and optimized to establish a better experimental method for two-dimensional electrophoresis (2-DE) analysis on the venom proteome of Conus betulinus Linnaeus native to Hainan. Molecular weights of the total proteins of Conus betulinus Linnaeus venom indicated most proteins located in three areas: 45-66.2 kD, 18.4-25 kD and under 14.4 kD. The PDI and other eight kinds of protein were isolated by 2-DE and confirmed by MALDI-TOF-MS successfully. Differential proteins in three different growing stages of Conus betulinus Linnaeus were studied and compared by 2-DE and PDQuest software, and five of them were confirmed by MALDI-TOF-MS. The technique of isolation and identification of venom protein by 2-DE and MALDI-TOF-MS was established, which would be the basis for isolating and purifying natural PDI from the venom of Conus betulinus and characterizing Conus snails venom proteomics.