"Proteases," or proteolytic enzymes, cleave the peptide bonds that join two amino acids. They adhere to the principles of hydrolytic reactions. Both prokaryotic and eukaryotic species manufacture proteolytic enzymes, which have important biological roles. Proteolytic enzymes are highly valuable commercially since the food, dairy, detergent, and leather processing industries all need them. A number of protease-based medicines have been approved, and proteolytic enzymes have also become recognized as therapeutics. Considering their great productivity and ease of enzyme purification, microorganisms constitute a key source of commercial proteolytic enzymes. Engineering new specificities, ensuring their stability, and using proteases as medicines provide significant scientific hurdles in the commercial utilization of proteases. In the current study, a bacterial stain with protease activity has been isolated. The production media has also been optimized using different carbon, nitrogen sources, metal ions, agricultural products, and physical parameters including temperature, pH, and incubation time. The soil samples were taken from the muralya dairy products pvt Ltd and creamline dairy products pvt Ltd Sankarankovil, Tenkasi . The isolated species was discovered to be a Bacillus species, according to the morphological and biochemical examinations. The isolated species' molecular identification was also completed using 16s RNA sequencing, and the results have been submitted to the gene bank under the accession numbers JKSP8 (OP835926.1) and JKSP9 (OP835912.). Using the BLAST similarity index, the two isolated strains JKSP8 and JKSP9 were recognized as Bacillus sp. and Bacillus velezensis, respectively. Nevertheless, optimizing large-scale protease manufacturing remains a difficult task. Thus, the main focus of this work was on the optimization and characterization of enzyme production with respect to pH, temperature, incubation duration, and various substrates or media for the manufacture of proteases, such as metal iron, carbon, nitrogen sources, and agro-bases. The optimization results reveal that the isolated organism JKSP9