The present context was investigated to isolate and characterize novel strain of industrially important bacteria from poultry farm. Preliminary study of poultry faeces sample was done by serial dilution followed by microscopic analysis, morphological characteristics and biochemical tests of pure isolated culture. The bacterium was identified as Bacillus species. Further, the identification of bacterium as Bacillus subtilis strain KPA was confirmed by subjecting its amplicon (483 bp) to 16S rRNA gene sequence analysis and pairwise alignment through BLAST tool. Minimum Free Energy (-192.10 kcal/mol), Mountain plot, Entropy, Partition, Pseudo Knots and Folds of the sequence were determined under RNA Secondary structure prediction through RNAeval Web server and RNAfold Web server. Percentage content of G+C (55.1%) was calculated through FTG server tool. Pictogram was visualized to identify alignment of the sequence. Phylogenetic tree based on taxonomic positions was inferred using the Neighbor-joining (NJ) algorithm in Molecular Evolution Genetic Analysis (MEGA) software version 4.0. The isolation of bacteria from different sources offers novel bacteria of unique functionality and their potential applications in different biotechnological processes. The present investigation clearly indicates the isolation, molecular characterization and sequence analysis (through Bioinformatics tools) of Bacillus subtilis strain KPA and its industrial applications.