2,6-Difluorobenzamide is an important intermediate with many applications in pesticide industries. Through screening a library of recombinant nitrile hydratases, the nitrile hydratase from Aurantimonas manganoxydans ATCC BAA-1229 was selected for production of 2,6-difluorobenzamide from 2,6-difluorobenzonitrile. Key parameters of the biocatalytic process, including temperature, pH, substrate loading, and substrate feeding mode, were optimized. Finally, 314g/L of 2,6-difluorobenzamide was produced in a simple batch process within 11h without formation of any by-product in an economical non-buffer system and similar result was obtained when scaled up to 30L. This study constitutes the first report of 2,6-difluorobenzamide significant production using a recombinant Escherichia coli-based biocatalyst.