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. We constructed various recombinant B. choshinensis strains co-expressing TK-PUL and molecular chaperone proteins. By screening for the extracellular enzymatic activity of TK-PUL, we identified the molecular chaperone proteins and corresponding recombinant B. choshinensis strains that optimized TK-PUL expression and secretion. Using the optimal strain co-expressing the chaperones, we optimized the fermentation conditions of recombinant B. choshinensis using the single-factor experiment method with response surface methodology. 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 strain 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 strain 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 represents 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.