The production of a recombinant nitrilase expressed in Escherichia coli JM109/pNLE was optimized in the present work. Various culture conditions and process parameters, including medium composition, inducer, induction condition, pH and temperature, were systematically examined. The results showed that nitrilase production in E. coli JM109/pNLE was greatly affected by the pH condition and the temperature in batch culture, and the highest nitrilase production was obtained when the fermentation was carried out at 37°C, initial pH 7.0 without control and E. coli was induced with 0.2 mM isopropyl-β-D-thiogalactoside at 4.0 h. Furthermore, enzyme production could be significantly enhanced by adopting the glycerol feeding strategy with lower flow rate. The enzyme expression was also authenticated by sodium dodecyl phosphate polyacrylamide gel electrophoresis analysis. Finally, under the optimized conditions for fed-batch culture, cell growth, specific activity and nitrilase production of the recombinant E. coli were increased by 9.0-, 5.5-, and 50-fold, respectively.