Pullulanase (PUL) plays a crucial role in breaking down α-1,6-glycosidic bonds in starch, a key process in starch processing and conversion. Based on PulB with high enzymatic activity, the expression of PUL in Bacillus subtilis was enhanced by plasmid screening, double promoter optimization, and signal peptide engineering. Furthermore, we innovatively employed a mussel foot protein to enhance the cell adhesion to carriers and utilized biofilm-based cell immobilization technology to optimize the fermentation process and stimulate biofilm formation. This approach led to a notably elevated enzyme activity, reaching 2233.56 U mL