Prediction of catalytic residues of an enzyme molecule is of great importance for a range of applications including molecular docking, drug design, structural identification and comparison of binding sites. Over the last decades, many studies have been conducted to identify the enzyme catalytic site. But, the catalytic residues of the azobenzene reductase from bacillus subtilis are still unknown. Investigation shows that under anaerobic conditions, azo dyes can be reduced by this enzyme and other environmental microorganisms to colorless amines, which may be toxic, mutagenic, and carcinogenic to humans and animals. To assess and estimate the toxicity, it is essential to identify the catalytic residues of this enzyme. The computational methods developed that address this issue are few. In this approach, we identify the catalytic residues of azobenzene reductase from bacillus subtilis, which were then analyzed in terms of properties including function, conservation, hydrogen bonding, B-factor, solvent accessibility, and flexibility. The results indicate that, Lys (83) and Tyr (74) play an important role as catalytic site residues in the azobenzene reductase from bacillus subtilis. It is hoped that this information will provide a better understanding of the molecular mechanisms involved in catalysis and a heuristic basis for predicting the catalytic residues in enzymes of unknown function. In this study, our approach mainly looks for a better understanding of the biodegradation of the Sudan I, Sudan II, Sudan III and Sudan IV dyes mediated by azobenzene reductase from bacillus subtilis. Further more, the catalytic site residues information is essential for understanding and altering substrate specificity and for the design of enzyme inhibitors.