Microbial cellulase enzyme, endoglucanase, performs a significant role in various industries, including textiles, paper pulp, biofuel production, agriculture, and pharmaceuticals. The aim of this research was focused on extracting, purifying, and investigating the microbial enzyme produced by Bacillus subtilis strain R2A (accession number PP088049). The cellulase-producing Bacillus strain was isolated from agricultural fields near Gwalior in Madhya Pradesh. The bacterial isolate of Bacillus species was preliminarily identified via cultural characteristics and biochemical characterization, using selective media culture. B. subtilis isolate exhibited significant cellulase enzyme activity under optimized conditions (pH 4.0-9.0, 37 °C, 120 rpm). The cellulase enzyme was refined via salting out, utilizing Diethylaminoethyl Cellulose-52 matrix column chromatography to purify microbial cellulase enzymes. The microbial cellulase was refined up to a 5.44 enrichment factor and had an catalytic activity of 137.95 U/mg protein. Microbial cellulase was a single-unit enzyme possessing an estimated protein molecular weight of 51.4 kDa, as evaluated through SDS-PAGE. This indicates the potential of B. subtilis isolate as a potent resource for biotechnological and industrial processes, particularly in producing sustainable fuels and cellulose-derived bioproducts. Microbial cellulases play an essential role in drug discovery by initiating the biotransformation of complex carbohydrates into secondary metabolites, as a result expediting the development of novel therapeutics, and improving medication efficacy. Future studies could focus on upscale production and exploring its application in diverse industrial settings.