The R-amidase production by a newly isolated strain of Delftia tsuruhatensis ZJB-05174 was optimized in this paper. Effects of factors such as carbon sources, nitrogen sources, and inducers on amidase production were investigated. The medium composition was optimized using central composite designs and response surface analysis. The optimal medium components for enhanced amidase production were found to be as follows: glucose, 8.23 g/l; yeast extract, 11.59 g/l; 2,2-(R,S)-dimethylcyclopropane carboxamide, 1.76 g/l; NaCl, 1 g/l; KH2PO4, 1 g/l; and K2HPO4, 1 g/l. A maximum enzyme production of 528.21 U/l was obtained under the optimized conditions, which was 4.7 times higher than that obtained under initial conditions.