The highly enantioselective epoxide hydrolase from Aspergillus niger is well utilized as biocatalysts for the preparation of enantiopure chiral epoxides and diols. Both growth of the fungus and EH activity production were found greatly affected by changing the carbon or the nitrogen source with fructose and corn steep liquor being the best. Their concentrations were optimized (10 g.l−1 of fructose and 15 g.l−1 of corn steep) which resulted in an increase of both the biomass produced (31%) and the epoxide hydrolase specific activity (38%). The results obtained suggested a complex regulation of the EH production. On the whole, a two times increase of the total EH activity was obtained.