Enzymes are vital biocatalysts facilitating biochemical reactions in organisms, playing pivotal roles in both biological systems and numerous industrial applications. This study focuses on the isolation and screening of asparaginase-producing bacteria from sewage water samples in Bhopal City. A modified M9 media, supplemented with asparagine and phenol red, was used to isolate asparaginase-producing bacteria. Out of the ten samples, seven exhibited asparaginase activity. Two bacterial strains, identified as Bacillus tropicus (S-1) and Priestiaveravalensis (S-2), were found to harbor the ansB gene. Sequence analysis revealed a 96.52% and 95.09% resemblance to Bacillus tropicus MCCC 1A01406 and Priestiaveravalensis strain SGD-V-76, respectively. These findings highlight the potential of these bacterial strains in producing L-asparaginase, emphasizing their significance in pharmaceutical applications, particularly in cancer treatment.