© ASABE 2021. All rights reserved.Distillers' dried grains with solubles (DDGS) is the co-product of dry-mill ethanol production and it has high nutritional value due to the residual fiber, protein, and lipid contents. The fiber content of this material can be used for production of hydrolytic enzymes such as cellulase and hemicellulases, which are needed to saccharify lignocellulosic biomass prior to ethanol fermentation. Although DDGS based media provides sugars and fibers for the enzyme production, the addition of media elements such as salts and nitrogen sources can further enhance enzyme production. Therefore, different salts such as KH2PO4, CaCl2·2H2O, MgSO4·7H2O, FeSO4·7H2O, CoCl2·6H2O, MnSO4·H2O, and nitrogen sources such as ammonium sulfate, peptone and yeast extract were evaluated for their effect on enzyme production in this study. Four fungal strains, three Aspergillus niger and one Trichoderma reesei were selected for evaluation of the effect of nutrients on enzyme production. Minerals did not have a significant effect on enzyme production (p<0.05). Ammonium Sulfate was a better nitrogen source as compared to peptone and yeast extract. Yeast extract improved the cellulase production (0.38 IU/ml) for A. niger (NRRL 1956) as compared to peptone (0.29 IU/ml). However, maximum cellulase productions of 0.42 IU/ml and 0.45 IU/ml were obtained by A. niger (NRRL 330) and A. niger (NRRL 567), respectively in presence of ammonium sulfate. Ammonium sulfate, peptone and yeast extract addition led to maximum hemicellulase production as 49.5, 42.49, 19.41 IU/ml by A. niger (NRRL 330), respectively. In conclusion, the results clearly demonstrated the positive effect of ammonium sulfate on cellulase production for two high enzyme producers.