For screening and isolation of microorganisms harboring nitrile-hydrolyzing enzymes that mediate the hydrolysis of iminodiacetonitrile (IDAN) to iminodiacetic acid (IDA), a sensitive and specific high-throughput screening model was established. This model integrated a solid screen-selective culture medium plate with bromcresol purple as the pH indicator coupled to Cu-IDA complex spectrophotometry. Four strains were selected to perform the biotransformation to IDA, which were isolated and identified as Alcaligenes faecalis, Pseudomonas chlororaphis, Pseudomonas putida and Klebsiella pneumoniae, on the basis of 16S rDNA sequence analysis in combination with physiological and biochemical characterization. Moreover, the maximum specific enzyme activity was 73.4 U/g dry cell weight obtained by A. faecalis ZJUTBX11 after optimization of the medium conditions for enzyme production. The results show that the proposed model is a suitable method for screening microorganisms with nitrile-hydrolyzing enzymes. We suggest the A. faecalis ZJUTBX11 strain to be used for large-scale bioconversion of IDAN to IDA, because of its excellent performance in the production of IDA.