The objective of this study was to evaluate the synthesis of xylanase enzyme by Bacillus safensis MABS6 using sorghum straw as a substrate. A comprehensive investigation was conducted to optimize xylanase yield by examining various parameters such as sorghum concentration, inoculum size, culture age, pH, temperature, and agitation speed. Additionally, the impact of nutritional additives, metallic ions, organic solvents, and alkaline H₂O₂ treatment on xylanase production and activity was explored. Experimental trials were performed with specific parameters, including 4% w/v sorghum concentration, 2% inoculum size, 12 hours of culture age, pH 7, 35°C, and 250 rpm. Further improvements involved the addition of nutritional additives such as gelatin, xylose, and potassium nitrate. The effects of initial conditions, various metallic ions (Ca²⁺, Mn²⁺, and Fe³⁺), and organic solvents (2-methyl propanol, methanol, and ethanol) on xylanase activity were evaluated. Additionally, saccharification investigations with a 4% alkaline H2O2 treatment assessed the enzymatic hydrolysis of sorghum straw. Optimized conditions resulted in a significant increase, with xylanase production reaching 3.49%. The partially purified xylanase exhibited over 60% relative activity within a pH range of 6 to 9 and demonstrated more than 55% activity between 45°C to 65°C after 1 hour. Certain metallic ions and organic solvents further enhanced xylanase activity. Notably, the xylanase derived from Bacillus safensis MABS6, utilizing sorghum straw, showcased desirable characteristics such as heat stability, alkali-solvent stability, and absence of cellulase activity. Its potential as a biocatalyst makes it valuable for various biotechnological applications, particularly in the efficient enzymatic hydrolysis of sorghum straw.