The culture conditions of recombinant E. coli harboring fungal nitrilase were systematically investigated in the level of shake flask, including medium composition, inducers, induction condition, pH and temperature. The results showed that a modified SOC medium containing trace elements was used as the fermentation medium with glycerol as the carbon source and peptone plus yeast extract as the nitrogen sources. The preferred inoculation amount was 0.5% (v/v). The induction for nitrilase production was carried out at 25°C and pH 7.5 with 0.5 mmol/L IPTG as the inducer for 12 h. The enzyme activity of the nitrilase were substantially improved through optimization and the total activity reached up to 3.8 U/mL, which was 3-fold higher than that of the initial level (0.84 U/mL). Subsequently, the nitrilase production in a 5-L stirred tank fermentor was determined and proved to be almost the same as that of shake flask. Preliminary study on biocatalytic properties of resting cells showed that the optimum reaction temperature and pH of the fungal enzyme were 45°C and pH 7.2.