To increase expression and extracellular secretion of pullulanase of the recombinant strain E.coli BL21(DE3)/pET20b(+)-BapulA at 3 L fermentor level. Expression of recombinant pullulanase in E. coli and further optimization of fermentation at the 3 L fermenter Firstly. The effects were investigaged, including the fermentation temperature, pH, IPTG concentration, induction time and glycine concentration on the expression of recombinant pullulanase. The optimum fermentation conditions were showed as followed: the growth temperature and pH of the pre-induction cells were 30 ℃ and 7.0, respectively. When the cell density reached 50 OD₆₀₀, the fermentation temperature was shifted to 25 ℃, and the pH was adjusted to 6.2 with lactose flow rates of 0.4 g/(L·h). During the fermentation process, glycine was added at the concentration of 1.5 g/L when the cell density of OD₆₀₀ reached 15, 45 and 75, respectively. When OD₆₀₀ reached 105, glycine was added at a concentration of 3 g/L. The optimal total and extracellular pullulanase activity were 1 910.1 U/mL and 659.0 U/mL, respectively, which represent 45.4 and 100-fold compared with those observed under initial conditions.