Bacterial polyphenol oxidase (PPO), also known as bacterial tyrosinase, is an enzyme present in some bacteria. This enzyme is similar in function to polyphenol oxidases found in plants and other organisms that oxidize phenolic compounds and is responsible for food browning, defensive mechanisms, and significant industrial applications. This study presents the optimized microbial production, extraction, and purification of PPO from Bacillus subtilis vk3, a strain isolated from Tirupur, India's wetlands. The enhanced PPO production was optimized through controlled conditions, considering factors like incubation time, pH, temperature, carbon source, nitrogen source, and substrate concentration through sequential experimentation. Enzyme production reached its peak at an incubation time of 42 h, a neutral pH of 7.0, and a temperature of 40°C. The enhanced PPO production was optimized through controlled conditions, considering factors like incubation time, pH, temperature, carbon source, nitrogen source, and substrate concentration through sequential experimentation. The enzyme yield reached its peak at an incubation time of 42 h, a neutral pH of 7.0 (111.52 U/ml), and a temperature of 40°C (114.69U/ml). Glucose (216.33 ± 2.08) and yeast extract (177 µg/ml) were identified as the most effective carbon and nitrogen sources, respectively. The optimal point for enzyme activity was determined to be a substrate concentration of 600mg/L. For purification, a combination of ammonium sulfate precipitation, dialysis, and gel filtration chromatography (Sephadex G-50) was employed. This process achieved a substantial increase in enzyme purity, with a noteworthy yield and specific activity. The molecular weight of the purified PPO was identified as 35 kDa. The study's findings offer valuable insights for industrial-scale PPO production from Bacillus subtilis vk3, highlighting its potential in various sectors. The research underscores the importance of optimizing production conditions to maximize yield and efficacy, setting a foundation for future applications and advancements in enzyme technology.