The main aim of this study was isolation of strains from different source like idly batter, dosa batter, raw milk and fresh water fish intestinal tract. Characterization of isolated bacterial culture was identified by morphological and 16s RNA molecular identification. Further it was selected for the evaluation of probiotic cation property using resistance to acid and bile tolerance, anti-microbial activity. It was rod-shaped, Gram-positive, catalase negative and produced ammonia from Arginine hydrolysis, which is the main characteristic feature, as observed with all lactobacilli. The culture could grow at different temperatures 30-37ºC, pH (5.8-6.2) and NaCl concentrations (2-12%) and in the presence of bile salt. The fish intestinal (FI) culture was similar to Lys. sphaericus strain L2 based on nucleotide homology and phylogenetic analysis. The FI sequence was producing 100% homology and significant alignments with taxa Lys. sphaericus strain L2. It was shown better growth even at room temperature hence this Lysinibacillus sphaericus was may be suggested used for probiotication of vegetable fruit juices.