Pullulanase is a debranching enzyme that cleaves explicitly alpha-1,6 glycosidic bonds, which is widely used in starch saccharification, production of glucose, maltose, and bioethanol. The thermal-resistant pullulanase is isolated from a variety of microorganisms; however, the lack of industrial production of pullulanase has hindered the transformation of the laboratory to industry. In this study, the expensive maltose syrup and soybean meal powder were replaced with cheap corn starch and corn steep liquor, exhibiting 440 U/mL of pullulanase in shake flasks by changing the C/N value and the total energy of the medium. Subsequently, the cultivation conditions were explored in a 50-L and 50-m(3)bioreactor. In batch culture, the pullulanase activity reached 896 U/mL, while it increased to 1743 U/mL in fed-batch culture by controlling the dissolved oxygen, pH, reducing sugar content, and temperature. Remarkably, the cultivation volume was enlarged to 50 m(3)based on the technical parameters of fed-batch culture. The industrial production of pullulanase was successful, and the activity achieved 1546 U/mL. When the product was stored at room temperature (25 degrees C) for 6 months, the pullulanase activity was over 90%. The half-lives at 60 and 80 degrees C were 119.45 h and 51.18 h, respectively, which satisfied the industrial application requirements of pullulanase.