Abstract: This study aimed to increase the expression and secretion of type III pullulan hydrolase (TK-PUL) by Brevibacillus choshinensis through the co-expression of molecular chaperone proteins and the optimization of fermentation conditions. By constructing multiple recombinant B. choshinensis co-expressing TK-PUL and molecular chaperone proteins, and screening them using extracellular enzyme activity as an indicator, the most favorable molecular chaperone protein and corresponding recombinant B. choshinensis for TK-PUL secretion and expression were determined. On this basis, single factor experiments and response surface methodology were used to optimize the fermentation conditions of recombinant B. choshinensis. Results showed that, the extracellular enzyme activity of the recombinant B. choshinensis strain co-expressing the molecular chaperone protein PrsA^Ba reached 98.79 U/mL, representing a 0.31-fold increase. The optimal medium for this recombinant B. choshinensis was composed of 19.65 g/L of glucose, 21.46 g/L of yeast extract, 12.01 g/L of MgCl₂·6H₂O, 9.02 g/L of proline, 0.01 g/L of FeSO₄·7H₂O, 0.01 g/L of MnSO₄·4H₂O, and 0.001 g/L of ZnSO₄·7H₂O. Culturing the optimized recombinant B. choshinensis in the above-mentioned optimized medium for 66 hours at 35 °C and an initial pH of 7.0 increased the extracellular TK-PUL activity to 192.68 U/mL, which represented a 1.56-fold increase. Efficient secretion of TK-PUL in B. choshinensis was achieved through the co-expression of molecular chaperone proteins and the optimization of fermentation conditions. This study provides a foundation for exploring the industrial-scale application of TK-PUL.