Alpha6beta2 nicotinic acetylcholine receptors (nAChRs) are potential therapeutic targets for the treatment of several neuropsychiatric diseases, including addiction and Parkinson's disease. Alpha-conotoxin (α-CTx) TxIB is a uniquely selective ligand, which blocks α6/α3β2β3 nAChRs only, but does not block the other subtypes. Therefore, α-CTx TxIB is a valuable therapeutic candidate peptide. Synthesizing enough α-CTx TxIB with high yield production is required for conducting wide-range testing of its potential medicinal applications. The current study optimized the cleavage of synthesized α-CTx TxIB resin-bounded peptide and folding of the cleaved linear peptide. Key parameters influencing cleavage and oxidative folding of α-CTx TxIB were examined, such as buffer, redox agents, pH, salt, co-solvent and temperature. Twelve conditions were used for cleavage optimization. Fifty-four kinds of one-step oxidative solution were used to assess their effects on each α-CTx TxIB isomers' yield. The result indicated that co-solvent choices were particularly important. Completely oxidative folding of globular isomer was achieved when the NH₄HCO₃ or Tris-HCl folding buffer at 4 °C contained 40% of co-solvent DMSO, and GSH:GSSG (2:1) or GSH only with pH 8~8.7.