This study covers the isolation, purification and characterization of polyphenol oxidase (PPO) from Bacillus subtilis, a bacterium of industrial prominence. The organism was cultured in nutrient broth supplemented with catechol under defined experimental conditions to induce PPO activity. Enzyme extraction involved sonication followed by acetone precipitation and column chromatography for purification. Maximum protein concentration (0.74 mg/mL) was witnessed with 60% acetone, while SDS-PAGE and HPLC analyses confirmed the enzyme's approximate molecular weight and retention time (20 min), respectively, aligning with previous findings. Enzyme activity was optimal at pH 4.0 and 40°C, with rapid decline observed outside this range. Kinetic analysis exhibited a Km of 55 mM for catechol, and enzyme inhibition by copper-chelating agents further verified its classification as a multicopper oxidase. The enzyme was successfully immobilized employing sodium alginate beads, enhancing its thermal and operational stability. Application trials in dough fermentation demonstrated improved rising ability in PPO-treated samples, suggestive of its potential role in bakery industries. Besides, data obtained from the present study provides new insights into the encouraging biotechnological applications of bacterial PPOs in biosensors, food processing and bioremediation.