Abstract: Neurospora crassa was used as the experimental strain,and the formula of high cell density medium was optimized using single factor experiment and response surface methodology. The optimum medium components were selected by the biomass of Neurospora crassa and were chosen as follows:Sucrose 42 g/L,peptone 36 g/L,KH₂PO₄ 2.6 g/L,ZnSO₄·7H₂O 0.038 g/L,MgSO₄·7H₂O 0.1 g/L,MnSO₄·H₂O 0.03 g/L,KCl 0.1 g/L,the verification experiment found that under the optimal medium composition,the maximum biomass of the bacteria was 11.295 g/L. In order to further improve the efficiency of high cell density culture,fed-batch culture was used to expand the biomass of Neurospora crassa. Results showed that the largest biomass of Neurospora crassa(14.732 g/L)based on the optimal medium was obtained by adding 10 mL of 200 g/L sucrose solution as the fed-batch culture at 24,60 and 96 h,respectively. The results provide theoretical support for the industrial application of Neurospora crassa.