A novel enantioselective amidase screening system was developed and proved to be efficient and accurate. This screening system employed acyl transfer activity of amidase in the presence of hydroxylamine, leading to the formation of hydroxamic acids, followed by spectrophotometric quantification of hydroxamic acid/iron(III) complexes. The enantioselectivities of amidase were evaluated by employing (R, S)-2, 2-dimethyl cyclopropanecarboxamide (1), (S)-2, 2-dimethyl cyclopropanecarboxamide and their mixture as substrates concurrently under the same conditions. To prove the accuracy of the screening system, enantioselectivity of acyl transfer reaction (E (T)) and that of hydrolytic reaction (E (H)) was compared. With this method, we obtained eight microorganism strains with enantioselective amidase from 523 isolates, two of which showed R-stereospecific avtivity for (R, S)-1.