To improve the yield of the Pseudomonas strain N1 in the fermentation culture medium screened from the biofilm of BAF reactor, the optimal shake flask fermentation conditions for Pseudomonas N1 were determined by single factor test and orthogonal test. The composition of optimal fermentation culture medium was as follows: nitrate of potash of 4 g/L, sodium succinate of 26.1 g/L, KH2PO4 of 4.0 g/L, MgSO4·7H2O of 1 g/L, KCl of 1 g/L. The cell concen- tration could reach 9.08 g/L under the conditions. Fermentation was performed for 36h in a 250ml shake flask containing 70mL medium at 1% inoculums volume and initial pH value 7.0. The cell concentration could reach 12.86 g/L which was 2.5 fold higher than that under the original conditions. Simultaneous nitrification and denitrification could be achieved to alleviate the pollution of nitrogen in the wastewater by adding the microbial inoculums into the common aerobic nitrifica- tion sludge.