Abstract: Objective To define the conditions for large-scale production of genetically engineered E.coli bearing humanized anti-HBsAg Fab. Method Characteristic growth and expression of the engineered E.coli were observed during fermentation in the shaking flask to define the optimal culture conditions to achieve the highest production levels. On the basis of the observation results, the E.coli was cultured in a fermentor using the fed-batch method to determine the optimal production techniques. Results Observation of the bacterium in the shaking flask showed initiation of the induction procedure in the mid-log growth phase at 25 ℃ with 0.2% arabinose resulted in the highest production of anti-HBsAg Fab. The D₆₀₀ value of the culture reached 55.2, equivalent to 110 g/L wet weight of the bacterium, using the DO-stat fed-batch method. The resultant Fab showed well-preserved biological activity. Conclusion Reliable techniques for rapid and massive production of the Fab have been developed.