Production of a thermostable pullulanase by DO‐stat fed‐batch fermentation of recombinant Escherichia coli BL 21 was investigated in a 5 L of fermentor. The effect of three oxygen control strategies, glucose feedback, shifting fermentor pressure, and adding oxygen‐enriched air, on cell growth and pullulanase expression were examined. The oxygen‐transfer capacity was found to be enhanced with increasing fermentor pressure and oxygen ratio in oxygen‐enriched air, but the cell growth and pullulanase production were restrained under high fermentor pressure. The highest cell density and pullulanase activity reached 55.1 g/L and 412 U/mL, respectively, in the case by adding oxygen‐enriched air, which was suggested as an effective approach to enhance both cell growth and pullulanase production. Practical applications This thermostable pullulanase displayed optimal activity at 90°C and pH 5.4, which could be applied for one‐step saccharification of starch biomass. The optimization of the DO‐stat fed‐batch fermentation in high cell density level would provide a research basis for its industrialization.