Nitrilase is one of the nitrile metabolizing enzymes that catalyses the conversion of nitriles to corresponding acids which has gained importance in green chemistry. Nitrilase being substrate specific yet it acts over a wide range of nitriles (aliphatic/aromatic) has drawn attention due to its utility in mild hydrolysis. Most of the nitrilases reported hitherto have been physically extracted characterized from the microbial/plant sources. In order to identify sequences for nitrilases two groups of motif were designed i.e. aliphatic nitrilase motif’s (MDMAl) and aromatic nitrilase motif’s (MDMAr) each with four motifs specifically belonging to nitrilase with conserved catalytic triad (Glu-48, Lys-131, Cys-165) which can be used as marker for nitrilase. Conserved regions were identified by performing Multiple Sequence Alignment (MSA) using Multiple EM for Motif Elicitation (MEME). The Manually Designed Motifs (MDM’s) were validated by ScanProsite and their presence is also confirmed by PRATT, Gblocks and MEME. The ScanProsite search against the MDMAr exhibited some new sources of aromatic nitrilase from plant, animals and microbes whereas MDMAl only exhibited nitrilase from microbes. Besides identifying unique motifs in order to confirm their substrate specificity for nitriles, randomly selected sequences were validated by studying some important physiochemical parameters and position specific amino acids.