Chemical synthesis has become an increasing popular approach for the production of proteins because of its flexibility and convenience in changing protein sequence and/or structure. However, at present, the efficiency of protein chemical synthesis is still low due to many methodological shortcomings. Here, we used a carbohydrate binding module (CBM) as a model molecule to explore the optimization of the oxidative folding of chemically synthesized proteins. By systematically comparing the reaction results, we were able to reveal the correlation between the overall folding rate and different reaction parameters, explain the possible reason for the observed correlation and identify the optimal conditions that maximize the synthetic efficiency. Overall, the findings from this study provided new insights into protein folding mechanisms and established new guiding principles for improving the efficiency of chemical protein synthesis.