Endo-1,4-β-xylanase (1,4-β-Xase) hydrolyzes hemicelluloses to xylose by the breakdown of linear polysaccharide chain β-1,4-xylan. The commercial applications of the enzyme include paper and pulp, bread and beverages, and ethanol production. The study aimed at optimizing the 1,4-β-Xase production of Bacillus pumilus by submerged fermentation using Plackett-Burman (P-B) design and central composite design (CCD). P-B design was used to screen the biotic variables and screened yeast extract, ferrous sulfate, manganese sulfate, and sugarcane bagasse as significant variables contributing the most to 1,4-β-Xase production. The R 2 value of the model was 1.0000 which indicated that the model is good. The screened four variables were optimized with CCD to study the interaction followed by optimization of abiotic variables (temperature and pH). Through CCD, yeast extract (10 g/L), ferrous sulfate (0.5 g/L), manganese sulfate (0.1 g/L), and sugarcane bagasse (50 g/L), temperature (37.5°C), and pH (7) were found to be the most significant factors affecting the 1,4-β-Xase production of B. pumilus. Our studies indicate the probability of highest production of 1,4-β-Xase on naturally available agro-waste sugarcane bagasse, which can be an alternative to standard substrate beechwood xylan for the production of 1,4-β-Xase.