This study explores the application of Response Surface Methodology (RSM) to optimize the physical conditions influencing the production of actinokinase, a fibrinolytic enzyme with significant therapeutic potential. Key parameters such as temperature, pH, agitation speed, and incubation time were systematically investigated to identify their effects on enzyme yield. A central composite design was employed to model and optimize the production process, resulting in enhanced actinokinase activity under ideal conditions. The results demonstrate that RSM is an effective statistical tool for fine-tuning critical physical factors, leading to a significant improvement in actinokinase production. These findings provide a foundation for scaling up enzyme production while maintaining cost-effectiveness and efficiency.