To increase the biomass and the activity ofEscherichia coliBL21(DE3) /pET-24a(+)-nhhBrbsArbsG(BAG) expressing high molecular mass nitrile hydratase fromRhodococcus rhodochrousJ1 and establish proper process of whole-cell catalysis of 3-cyanopytidine and high cell-density cultivation, we used orthogonal experiment (L9(34)) to optimize elements in culture media, comparing methods of substrate-flow and substrate fed-batchand applying technology of fed-batch to achieve high cell-density cultivation. After the optimization of culture media, the biomass and the specific activity of BAG reached4.42 gDCW/L and 30.91 U/mgDCW in the flask, respectively. The process of substrate fed-batch was more appropriate and when it was applied in the production of nicotinamide using BAG, the concentration of nicotinamide reached 390 g/L. After the high cell-density cultivation of BAG in 5 L bioreactor, the biomass of cell attained 73.97 gDCW/L(OD600=200), the specific activity and the total activity reached 42.70 U/mgDCW and 2813 U/mL, respectively. When we used high density of BAG after fermentation in the tank to catalyze 3-cyanopytidine, we obtained 537?g/L of nicotinamide, which is the highest concentration of nicotinamide that produced in recombinantE.coilexpressing nitrile hydratase.