The present study deals with the production of alkaline and thermostable polygalacturonase from the isolated soil bacterium Bacillus subtilis (C4) through submerged fermentation. In this study, a total of 66 bacterial colonies were isolated from six sampling sites. Of these, eight isolates exhibited polygalacturonase activity, with the C4 isolate showing the highest activity. The potential analysis of the new isolate was conducted in comparison to three other polygalacturonase-producing microorganisms, with isolate C4 demonstrating the maximum enzyme activity and therefore selected for further investigation. The characterization and identification of isolate C4 as Bacillus subtilis C4 were carried out, and the growth profiles were analyzed under submerged fermentation. The results showed that the organism reached maximum log phase by the 24th hour, followed by a gradual reduction in growth, which remained stable until the 72nd hour, indicating a stationary phase at 40 °C and pH 11.0. Various process parameters such as incubation time, temperature, pH, inoculum volume, inoculum age, and agitation were investigated to achieve maximum polygalacturonase production.